1. home and garden

In vitro propagation of “Sarra” rose cv.
Rosa sp. using axillary buds.
By
Sarra Ali Saad Hamed
B.Sc. (Honours)
Faculty of Agriculture
Kharoum University
1999
A Thesis submitted in Partial Fulfillment of
The Requirement of the Degree of Master
of Science in Agriculture
Thesis superviser: Prof.Abd El Gaffar El Hag Saeed .
Thesis co- superviser: Dr.Sayda Omer El Howayris .
Department of Agricultural
Biotechnology and Botany
University of Khartoum
Sudan(2003)
Dedication
To all my dignified teachers and virtuous people who
encouraged me.
To my great dearest teachers father and mother who tought me
loving
knowledge and Perseverance .
To my dearest husband who was very helpfull and patient with me .
To my dearest sister Sawsan and all my relatives and friends who
helped me so much .
To my dearest sun Ali who tought me the meaning of life .
I give them this research as a present with all my great love and
respect .
Sarra
Acknowledgement
Praise and thanks to Allah who enabled me to finish this
research .
I would like to thank all those who helped me to carry out
this research and bring it to its last form .
My great thanks to my helpfull supervisor prof.Abd El
Gaffar El Hag Said for all his great help in all this research stages. I
am greatfull for all his time , knowledge and experience which he
shared with me generously.
Also I would like to express my gratitude to Dr. Sayda omer
El Howayris my co-supervisor who spared no effort to supervise
and guide this study. Iam great full for her encouragement, advice,
and patience .
I would like to express my gratitude to all Leena company
staff who were very helpful and patient with me until I finished my
practical work .
All my thanks for all my friends, relatives, and all virtuous
people who helped me to finish this study and bring it in this form .
Contents
1- Introduction
1
2- Literature Review:
2-1.Botany of Roses
2-2.Taxonomy
2-3.Uses of Roses
2-4.Propagation of Roses
2-4.1.Traditional Techniques
2-4.2. Tissue Culture Techniques
2-4.2.1.Media Used
2-4.2.2.Plant Material and Explantation
2-4.2.3.Preparation of Media
2-4.2.4.Preparation of Explants
2-4.2.5.Tissue Culture of higher Species
2
2
2
4
6
6
7
7
8
8
8
9
3- Materials and Methods:
3-1.Plant Material
3-2.Sterilization
3-3.Culture Media
3-4.Culture of Explants
3-5.Experimentations
3-5.1.The Effect of Different Concentrations of Murashige and Skoog
(1962)Salt Mixture on In Vitro Growth and Development of
rose Axillary Buds.
3-5.2. The effect of Different Concentrations of Sodium
Hypophosphate Salt(NaH2PO4) on In Vitro Growth
and Development of rose Axillary Buds.
3-5.3.Energy Source: (Carbohydrates):
3-5.3.1.Sucrose
3-5.3.2.Glucose
3-5.4. The Effect of Different Concentrations of ThiamineHCl (Vitamin B1) on In Vitro Cultured Rose Axillary Buds
3-5.5. The Effect of Different Concentrations of Myo-Inositol
on In Vitro Cultured Rose Axillary Buds
3-5.6. The Effect of Different Concentrations of Adenine Sulphate
(A/S) on In Vitro Cultured Rose Axillary Buds
3-5.7. The Effect of Different Concentrations of Naphthalene Acetic
Acid (NAA) and Benzyle Adenine(BA) on In Vitro Growth
Rose Axillary Buds
18
18
18
18
18
18
18
20
20
20
20
20
20
20
20
3-5.8. The Effect of the Physical state of the Nutient Medium on
the growth and development of In Vitro Cultured Rose
Axillary Buds
3-5.9. The Effect of Darkness on growth and development
of In Vitro Cultured Rose
3-6.Statistical Analysis
20
20
20
4-Results :
22
4-1.Effect of different concentrations ofMurashige and Skoog
(MS,1962) salt mix on growth and development of rose axillary buds
22
4-2. Effect of different concentrations of sodium hypophosphate
NaH2PO4 salt on growth and development of rose axillary buds
22
4-3.Effect of energy sources(carbohydrates):
4-3.1. Effect of different concentrations of sucrose on growth
and development of rose axillary buds
4-3.2. Effect of different concentrations of glucose on growth
and development of rose axillary buds
22
22
4-4.Effect of different concentrations of Thiamine –HCl
(Vitamin B1 )on growth and development of rose axillary buds
37
4-5. Effect of different concentrations of Adenine Sulphate (A/S)
(Vitamin B1 )on growth and development of rose axillary buds
37
4-6. Effect of different concentrations of myo-inositole
on growth and development of rose axillary buds
37
4-7. Effect of different concentrations of growth regulators :
4-7.1.Naphthaline acetic acid (NAA)
4-7.2.Benzyl adenine(BA)
4-7.3. Benzyladenine(BA) + Naphthaline acetic acid (NAA)
37
37
37
57
4-8. Effect of the physical state of the medium
57
4-9.The effect of darkness
57
5-Discussion :
69
5-1. Effect of different concentrations of (MS) salt mix
on growth and development of rose axillary buds
69
22
5-2. Effect of different concentrations of sodium hypophosphate
NaH2PO4 salt on growth and development of rose axillary buds
5-3. .Effect of energy sources(carbohydrates)
5-3.1. Effect of different concentrations of sucrose on growth
and development of rose axillary buds
5-3.2. Effect of different concentrations of glucose on growth
and development of rose axillary buds
69
69
69
70
5-4. Effect of different concentrations of Thiamine –HCl
(Vitamin B1 )on growth and development of rose axillary buds
70
5-5. Effect of different concentrations of Adenine Sulphate (A/S)
(Vitamin B1 )on growth and development of rose axillary buds
70
5-6. Effect of different concentrations of myo-inositole
on growth and development of rose axillary buds
71
5-7. Effect of different concentrations of growth regulators on
growth and development of axillary buds
5-7.1. Naphthaline acetic acid (NAA)
5-7.2. Benzyl adenine(BA)
5-7.3. Benzyl adenine(BA) + Naphthaline acetic acid (NAA)
71
71
71
71
5-8. Effect of the physical state of the medium on growth and
development of rose axillary buds
72
5-9.Effect of darkness
72
6-References
7- Appendix (1)
8- Appendix (2)
74
86
87
List of Tables
Table(1): Effect of different concentrations of MS salts on in vitro
growth and development of rose axillary buds.
23
Table(2): Effect of different concentrations of NaH2PO4 salts on
in vitro growth and development of rose axillary buds.
26
Table(3): Effect of different concentrations of sucrose on
in vitro growth and development of rose axillary buds.
29
Table(4): Effect of different concentrations of glucose on
in vitro growth and development of rose axillary buds.
33
Table(5): Effect of different concentrations of thiamine -HCl on
in vitro growth and development of rose axillary buds.
38
Table(6): Effect of different concentrations of adenine sulphate on
in vitro growth and development of rose axillary buds.
42
Table(7): Effect of different concentrations of myo-inositol on
in vitro growth and development of rose axillary buds.
46
Table(8): Effect of different concentrations of NAA on
in vitro growth and development of rose axillary buds.
49
Table(9): Effect of different concentrations of BA on
in vitro growth and development of rose axillary buds.
53
Table(10): Effect of different concentrations of BA+NAA on
in vitro growth and development of rose axillary buds.
58
Table(11): Effect of the physical state of the medium on the
in vitro growth and development of rose axillary buds.
62
Table(12): Effect of darkness on in vitro growth and development
of rose axillary buds.
66
List of Figures
Fig.(1): Effect of MS saltsmix concentration on number of
leaves of in vitro cultured plantlets of “Sarra” rose cultivar
24
Fig.(2): Effect of different concentrations of NaH2PO4 salt on plant
height(cm) of in vitro cultured plantlets of “Sarra” rose cultivar
27
Fig.(3): Effect of different concentrations of NaH2PO4 salt on number
of leaves of in vitro cultured plantlets of “Sarra” rose cultivar
28
Fig.(4): Effect of different concentrations of sucrose on plant
height(cm) of in vitro cultured plantlets of “Sarra” rose cultivar
30
Fig.(5): Effect of different concentrations of sucrose on number
of leaves of in vitro cultured plantlets of “Sarra” rose cultivar
31
Fig.(6): Effect of different concentrations of glucose on number
of shoots of in vitro cultured plantlets of “Sarra” rose cultivar
34
Fig.(7): Effect of different concentrations of glucose on number
of nods of in vitro cultured plantlets of “Sarra” rose cultivar
35
Fig.(8): Effect of different concentrations of thiamine-HCl on plant
height(cm) of in vitro cultured plantlets of “Sarra” rose cultivar
39
Fig.(9): Effect of different concentrations of thiamine-HCl on number
of shoots of in vitro cultured plantlets of “Sarra” rose cultivar
40
Fig.(10):Effect of different concentrations of adenine sulphate on number
of leaves of in vitro cultured plantlets of “Sarra” rose cultivar
43
Fig.(11):Effect of different concentrations of adenine sulphate on number
of shoots of in vitro cultured plantlets of “Sarra” rose cultivar
44
Fig.(12): Effect of different concentrations of myo-inositol on number
of leaves of in vitro cultured plantlets of “Sarra” rose cultivar
Fig.(13): Effect of different concentrations of myo-inositol on plant
height(cm)of in vitro cultured plantlets of “Sarra” rose cultivar
Fig.(14): Effect of different concentrations of NAA on plant
height(cm)of in vitro cultured plantlets of “Sarra” rose cultivar
47
48
50
Fig.(15): Effect of different concentrations of NAA on number of roots
of in vitro cultured plantlets of “Sarra” rose cultivar
51
Fig.(16): Effect of different concentrations of BA on plant
height(cm)of in vitro cultured plantlets of “Sarra” rose cultivar 54
Fig.(17):Effect of different concentrations of BA on number
of shoots of in vitro cultured plantlets of “Sarra” rose cultivar
55
Fig.(18): Effect of different concentrations of BA+NAA on plant
height(cm)of in vitro cultured plantlets of “Sarra” rose cultivar 59
Fig.(19): Effect of different concentrations of BA+NAA on number
of leaves of in vitro cultured plantlets of “Sarra” rose cultivar
60
Fig.(20): Effect of the physical state of the medium on plant height
(cm)of in vitro cultured plantlets of “Sarra” rose cultivar
63
Fig.(21): Effect of the physical state of the medium on number of
leaves of in vitro cultured plantlets of “Sarra” rose cultivar
64
Fig.(22): Effect of darkness on plant height (cm)of in vitro
cultured plantlets of “Sarra” rose cultivar
67
Fig.(23): Effect of darkness on number of nods of in vitro
cultured plantlets of “Sarra” rose cultivar
68
List of Plates
Plate(1): Effect of the three multiplication media (A ,B ,and C) on
the growth of the explants of “Sarra” rose.
19
Plate(2): “Sarra” rose plantlets growing on different MS salt mix
strengths .
25
Plate(3): “Sarra” rose plantlets growing on different concentrations
of sucrose .
32
Plate(4): “Sarra” rose plantlets growing on different concentrations
of glucose .
36
Plate(5): “Sarra” rose plantlets growing on different concentrations
of thiamine –HCl .
41
Plate(6): “Sarra” rose plantlets growing on different concentrations
of adenine sulphate.
45
Plate(7): “Sarra” rose plantlets growing on different concentrations
of NAA .
52
Plate(8): “Sarra” rose plantlets growing on different concentrations
of BA.
56
Plate(9): “Sarra” rose plantlets growing on different concentrations
of BA +NAA .
Plate(10): “Sarra” rose plantlets cultured on agar or cotton .
61
65
Abstract
This study was carried out at Lena Plant Tissue Culture Laboratory
(Um al-kora) to determine the optimum concentration of Murashige and
Skoog(MS, 1962) medium components for the growth of “Sarra” rose
cultivar.
Axillary buds were grown on Murashige and skoog multiplication
medium (A). several trails were then conducted to modify his medium to
fit “Sarra” rose cultivar’s clonal propagation.
Full strength MS salt mix containing 30 gm/l sucrose, full strength
sodium phosphate(Na H2 PO4) ,2.0 X Thiamine-HCL),and (0.3 mg/l)
benzyl adenine (BA) for shoot growth and development . best rooting was
obtained when using the above medium components plus 0.3 mg/l
naphthalene acetic acid (NAA) instead of BA. Adding each growth
regulator alone gives better results than adding them in combination.
Adding adenine sulphate and myo-inositol to the medium doesn’t
make great differences in plant growth but they improve it to some
extent.
Glucose can be used instead of sucrose and it gives better results
than sucrose but the problem is that, glucose is more expensive than
sucrose so it is not economically feasible.
Using cotton instead of agar gave excellent results which
encourage using it to avoid the expensive cost of agar.
Incubation of plantlets under darkness did not give good results except
plant height so there is no need for this treatment except if there is an
urgent need for a quick
increase in plant height.
‫ﻣﻠﺨﺺ ﺍﻻﻃﺮﻭﺣﺔ‬
‫ﺃﺟﺮﻯ‪ ‬ﻫﺬﺍ ﺍﻟﺒﺤﺚ ﺑﻤﻌﻤﻞ ﺷﺮﻛﺔ ﻟﻴﻨﺔ ﻟﺰﺭﺍﻋﺔ ﺍﻻﻧﺴﺠﺔ‬
‫ﺍﻟﻨﺒﺎﺗﻴﺔ ﺍﻟﻤﺤﺪﻭﺩﺓ ﺑﺄﻡ ﺍﻟﻘﺮﻯ‪ ،‬ﻻﻳﺠﺎﺩ ﺍﻟﺘﺮﺍﻛﻴﺰ ﺍﻟﻤﺜﺎﻟﻴﺔ ﻟﻮﺳﻂ ﻣﻮﺭﺍﺷﻴﺠﻰ‬
‫ﻭﺳﻜﻮﻕ ‪1962‬ﻡ ﺍﻟﺼﺎﻟﺤﺔ ﻟﻨﻤﻮ ﺻﻨﻒ ﺍﻟﻮﺭﺩ ))ﺳﺎﺭﺓ((‪.‬‬
‫ﺯﺭﻋﺖ ﺍﻟﺒﺮﺍﻋﻢ ﺍﻟﺠﺎﻧﺒﻴﺔ ﻓﻰ ﻭﺳﻂ ﻣﻮﺭﺍﺷﻴﺠﻰ ﻭﺳﻜﻮﺝ )ﺃ(‬
‫ﻭﺍﻋﻘﺐ ﺫﻟﻚ ﻋﺪﺓ ﺗﺠﺎﺭﺏ ﻟﺘﺤﻮﻳﺮ ﻫﺬﺍ ﺍﻟﻮﺳﻂ ﻟﻴﻼﺋﻢ ﺍﻟﺘﻜﺎﺛﺮ ﺍﻟﺴﻼﻟﻰ ﻟﺼﻨﻒ‬
‫ﺍﻟﻮﺭﺩ )ﺳﺎﺭﺓ(‪.‬‬
‫ﺃﻓﻀﻞ ﺑﻴﺌﺔ ﻟﻨﻤﻮ ﻭﺗﻜﺸﻒ ﺍﻷﻓﺮﻉ ﻫﻰ ﺃﻣﻼﺡ ﻣﻮﺭﺍﺷﻴﺠﻰ ﻭﺳﻜﻮﻕ‬
‫ﺍﻟﺨﻤﺴﺔ ﻋﻨﺪ ﺍﻟﺘﺮﻛﻴﺰ ﺍﻟﻜﺎﻣﻞ ﻷﻣﻼﺡ ﻫﺬﺍ ﺍﻟﻮﺳﻂ ﻣﻊ‪30،‬ﻣﻠﺠﻢ‪/‬ﻟﺘﺮ ‪،‬ﺳﻜﺮﻭﺯ‬
‫( ( ﺿﻌﻒ ﺗﺮﻛﻴﺰ ‪Na H2 PO4‬ﻭﺍﻟﺘﺮﻛﻴﺰ ﺍﻟﻜﺎﻣﻞ ﻟﻤﻠﺢ ﻓﻮﺳﻔﺎﺕ ﺍﻟﺼﻮﺩﻳﻮﻡ‬
‫(( ﺗﻢ ﺍﻟﺤﺼﻮﻝ ﻋﻠﻰ ‪BA‬ﻓﻴﺘﺎﻣﻴﻦ ﺍﻟﺜﻴﺎﻣﻴﻦ ﻣﻊ ‪ 0.3‬ﻣﻠﺠﻢ‪/‬ﻟﺘﺮ ﺑﻨﺰﻳﻞ ﺍﺩﻳﻨﻴﻦ‬
‫ﻟﻠﻮﺳﻂ ‪NAA‬ﺍﻓﻀﻞ ﺗﺠﺬﻳﺮ ﺑﺎﺿﺎﻓﺔ ‪ 0.3‬ﻣﻠﺠﻢ‪/‬ﻟﺘﺮ ﻣﻦ ﺣﻤﺾ ﻧﺎﻓﺘﺎﻟﻴﻦ ﺍﻟﺨﻠﻴﻚ‬
‫ﺍﻟﻐﺬﺍﺋﻰ ﺍﻋﻼﻩ ﺑﺪﻻﹰ ﻣﻦ ﺍﻟﺒﻨﺰﺍﻳﻞ ﺍﺩﻳﻨﻴﻦ ﻭﻛﺎﻧﺖ ﺍﺿﺎﻓﺔ ﻛﻞ ﻫﺮﻣﻮﻥ ﻋﺎﻯ‬
‫ﺣﺪﺓ ﺗﻌﻄﻰ ﻧﺘﺎﺋﺞ‬
‫ﺃﻓﻀﻞ ﻣﻦ ﺍﺿﺎﻓﻨﻬﺎ ﻣﻊ ﺑﻌﻀﻬﺎ ﻟﻠﻮﺳﻂ ‪.‬‬
‫ﺍﺩﺕ ﺍﺿﺎﻓﺔ ﻛﺒﺮﻳﺘﺎﺕ ﺍﻟﺪﻳﻨﻴﻦ ﻭﻣﻴﻮﺃﻧﻮﺳﻴﺘﻮﻝ ﻟﺘﺤﺴﻦ ﺍﻟﻨﻤﻮ‬
‫ﺑﻌﺾ ﺍﻟﺸﻰ ﻭﻟﻜﻦ ﻟﻢ ﺗﻄﻔﻰ ﻓﺮﻭﻗﺎﺕ ﻣﻌﻨﻮﻳﺔ ﻋﻠﻴﻪ‪.‬‬
‫ﻳﻤﻜﻦ ﺍﺳﺘﺨﺪﺍﻡ ﺳﻜﺮ ﺍﻟﺠﻠﻜﻮﺯ ﻛﺒﺪﻳﻞ ﻟﺴﻜﺮ ﺍﻟﺴﻜﺮﻭﺯ ﺣﻴﺚ‬
‫ﺗﻌﻄﻰ ﻧﺘﺎﺋﺞ ﺍﻓﻀﻞ ﻣﻨﻪ ﻟﻜﻦ ﻳﻌﺎﺏ ﻋﻠﻴﻪ ﺗﻜﻠﻔﺘﺔ ﺍﻟﻌﺎﻟﻴﺔ ﻣﻘﺎﺭﻧﺔ ﻣﻊ ﺍﻟﺴﻜﺮﻭﺯ‬
‫ﺣﻴﺚ ﻳﺼﺒﺢ ﺍﺳﺘﻌﻤﺎﻟﻪ ﻏﻴﺮ ﺍﻗﺘﺼﺎﺩﻯ ‪.‬‬
‫ﺍﺳﺘﺨﺪﺍﻡ ﺍﻟﻘﻄﻦ ﺑﺪﻝ ﺍﻷﺟﺎﺭﺍﻋﻄﻰ ﻧﺘﺎﺋﺞ ﻣﻤﺘﺎﺯﺓ ﻣﻤﺎ ﻳﺸﺠﻊ‬
‫ﺍﻣﻜﺎﻧﻴﺔ ﺍﺳﺘﻌﻤﺎﻟﻪ ﻛﺒﺪﻳﻞ ﻣﺜﺎﻟﻰ ﻟﻸﺟﺎﺭ ﻟﺘﺤﺎﺷﻰ ﺗﻜﻠﻔﺔ ﺳﻌﺮ ﺃﻻﺟﺎﺭ ﺍﻟﺒﺎﻫﻈﺔ‪.‬‬
‫ﺣﻀﺎﻧﺔ ﺍﻟﻨﺒﻴﺘﺎﺕ ﻓﻰ ﺍﻟﻈﻼﻡ ﻟﻢ ﺗﻌﻄﻰ ﻧﺘﺎﺋﺞ ﺟﻴﺪﺓ ﻋﺪﺍ ﺍﻟﻄﻮﻝ ﻟﺬﺍ‬
‫ﻻﻳﻨﺼﺢ ﺑﻬﺬﻩ ﺍﻟﻤﻌﺎﻣﻠﺔ ﺍﻻ ﻓﻰ ﺣﺎﻟﺔ ﺍﻟﺤﻮﺟﺔ ﺍﻟﻤﻠﺤﺔ ﻻﻋﻄﺎﺀ ﺍﻻﺳﺘﻄﺎﻟﺔ ﺍﻟﺴﺮﻳﻌﺔ‬
‫ﻟﻠﻨﺒﺎﺕ ‪.‬‬
Abreviations
NAA: αnaphthalene acetic acid.
Kin/kn:Kinetin.
BA/BAP: Benyl adenine/Benzyl aminopurine
IBA:Indol butyric acid.
IAA:Indole acetic acid.
2iP:2-Isopentyl adenine.
2, 4-D:2,4-Dichorophenoxy acetic acid.
GA3:Gibberellic acid.
BPA:N-benzyl-9(2 tetrahydro Pyranl) adenin
TDZ:N-Phynyl-N-1,2,3-thidiazol-5 ylurea.
MS:Murashige and skoog(1962).
B5:Gamborg, et al.(1968).
GD:Gresshoff and Doyl.
SH:Schenk and Hild Brandt
WS:Wolter and Skoog.
WPM:Lloyd and Mc Cown Woody Plant Medium.
NaH2PO4:Sodium hypophosphate.
expt.:experiment.
h/hr:hour.
Min:minute.
Conc.:concentration.
1/ Introduction
Roses are grown for their profusion of flowers (often fragrant),and
sometimes for their fruits. Roses have been cultivated as garden plants since very
early times.
They are the most widely grown and loved of the flowering woody
perennials. Commercially , they are probably the most important flower crop in the
world. But till now very little is known about their culture.
In Sudan Roses are the most popular ornamental plants . They are grown
in garden beds or in pots for general landscape purposes.
There has been a real consiousness about the importance of landscaping
and ornamental plants in varios towns of the country the last few years indicating
high rate of urbanization. On the other hand , roses can be considered as a cash by
crop exporting cut rose flowers during the winter time especially to Europe.
Very few researchs has been done on rose culture in Sudan in general and
tissue culture in particular . One of the most important roses in Sudan is a variety
called “Sarra” . This cultivar bears high temperature and it flowers all the year round
even during the summer while the other varieties flower only during the winter
months.
Sarra like other rose cultivar is traditionally propagated by budding .This
method of clonal propagation is tedious laborious and is very slow.
Trials to use tissue culture in “Lena Company” for the clonal propagation of “Sarra”
were not successful. This research was carried out as an attempt to tailor the medium
components that will best suit the clonal propagation of “Sarra”, the most expensive
rose among all other rose cultivars
2/ Literature Review
2-1 Botany of roses:
Roses belong to the family Rosaceae. Plants of the genus Rosa are deciduos or
sometimes evergreen shrubs,with an up right, climbing or trailing stems, usually
prickly, rarely un- armed .Leaves are divided into usually 5to7 oval leaflets,with
rounded or pointed tips, that are sometime toothed. Stems usually bear thorns , or
prickles fully and hardy. Roses are polycarpic self induuctive plants , which initiate
flowers irrespective of photoperiod or temperature. They initiate flowers
autonomously on every growing shoot after a certain size is attained. Every shoot
blooms unless the flower bud dies early in its development. Roses have a determinant
inflorescence that may assume a corymbose , paniculate , or solitary form. When
flowers are borne singly, as in many hybrid tea cultivars, there are still undeveloped
flower buds in the axils of the leaves immediately below the terminal flower. These
buds can develop into short flowering shoots under favorable environmental
conditions. Flowers are borne on an upright , prickly stems (the end of short
branchlets). Sepals and petals are 5 , rarely 4 , stamens and pistils are numerous ,
enclosed in a usually urnshaped receptacle. Flower colours range from, white , pink ,
yellow , orange , to lavender with many shades , hues and tints in between. The
doubling of a flower is simply the replacement of the stamens and styles by petals
Fruits formed from fertilized flowers are called hips which become fleshy and berrylike at maturity , enclosing several or many bone achenes. They are reputed to be high
in vitamin C content and consequently are in high demand by health food advocates
(Mohamed,1994;Larson,1980; Brickell,1989).
2-2 Taxonomy:
Present day commercial rose cultivars are all hybrids of rose species that are
extinct. Even the earlier representations are probably a long way from the wild
species.
(Larson,1980);Gault and Synge,1971)
Wild roses and hybrid species share most of the characteristics of the parent species
including shrubs and climbing roses. Today varieties of Roses are divided into old
garden roses and modern garden roses.
2.2.1Old garden roses :
2.2.1.1.Alba:
large freely branching shrubs with clusters of usually 5-7 semi.double to double
flowers.
2.2.1.2.Bourbon:
Open remontant shrubs that may be trained to climb produces usually fully double
flowers.
2.2.1.3.China:
Spindly remontant shrubs that produce single to double flowers.
2.2.1.4.Damask:
Open shrubs with usually very fragrant semi-to fully double flowers.
2.2.1.5.Gallica:
Shrubs of fairly dense free-branching growth.
2.2.1.6.
Hybrid Perpetual:
Vigorous, free branching , remontant shrubs that bear fully double flowers.
2.2.1.7.Moss:
Ofen lax shrubs with a fury moss-like growth on stems and calyex.
2.2.1.8.Noisette:
Remontant climbing roses that bear clusters of up to 9 usually double flowers with a
slight spicy fragrance.
2.2.1.9.Portland:
Upright remontant shrubs with simi-double to double flowers.
2.2.1.10.Provence:
Thorny shrubs that produce scented , usually double to fully double flowers.
2.2.1.11.Sempervirens:
Semi ever green climbing roses with numerous semi to fully double flowers.
2.2.1.12.Tea:
Remontant shrubs and climbing roses that produce spicy scented , stander – stemmed
, pointed , semi- to fully double flowers.
2.2.2. Modern roses:
2.2.2.1.Shrub:
A diverse group of modern roses, most of which are remontant , that grow larger
(mostly 1-2 meters height) than most bush roses. Has single to fully double flowers ,
held singly or in sprays , in summer and / or autumn. It is suitable for beds and
borders and for growing as specimen plants.
2.2.2.2.Large flowered bush (Hybrid Tea):
Remontant shrubs with mostly pointed , double flowers , (8cm) or more across , borne
singly or in groups of 3 ,it flowers in summer- autumn and is excellent for beds,
borders, hedges and for cutting.
2.2.2.3.Cluster flowered bush (Floribunda):
Remontant shrubs that produce sprays of usually 3-25 single to fully double flowers
in summer – autumn. It is excellent for beds, borders, hedges and for cutting. “Sarra”
roses belong to this group.
2.2.2.4.
Dwarf cluster flowered bush (Patio):
Neat, remontant shrubs , 38-60 cm high, that – bear sprays of generally 3-11 single to
double flowers in summer autumn. It is ideal for beds, borders, hedges and for
growing in containers.
2.2.2.5.
Miniature bush:
Remontant shrubs , 45 cm high , with sprays of usually 3-11 tiny , single to fully
double flowers in summer-autumn. It has tiny leaves . It is suitable for rock gardens ,
small spaces and for growing in containers.
2.2.2.6.
Polyanthe:
Tough, compact, remontant shrubs with sprays of usually 7-15 small 5 petalled ,
single to double flowers.It flower in summer – autumn. Its suitable for beds.
2.2.2.7.
Ground cover:
Trailing and spreading roses , many of them are remontant with single to fully double
flowers , borne mostly in clusters of 3 – 11 and flower in summer and / or autumn.
They are suitable for beds , banks and walls.
2.2.2.8.
Climbing :
Vigrous climbing roses , some of which are remontant , with stiff stems and
borne single to fully double flowers . They are borne singly or in clusters from late
spring to autumn. They are suitable for training over walls fences and pergolas.
2.2.2.9.
Rambler:
Vigorous climbing roses with lax stems. They have clusters of 3-21 single to fully
double flowers .Flowering mainly in summer and are suitable for training over walls ,
fences , pergolas and trees.
2.3.
Uses of Roses :
2.3.1. Wild life:
Roses hips are eaten by migration bird and used as a winter food for song birds .
Rabbits feed on the bark and the shoots . Wild roses form thickets used as nesting
sites for small mammals and birds.
2.3.2. Horticulture:
Roses are used in landscaping cover and hedges. They are also used as cut
flowers. Roses are available in almost any color imaginable and are suited to a
number of sites.
2.3.3. Medicinal:
Fruits are eaten as a source of vitamin C and are used to treat diarrhea. Bark tea
is also used for dysentery. Flowers are used in China by some people as a stimulant
and as a tonic .They promote and Improve blood circulation,and reduce stomachache.
Decoration of the bark is used to treat worms.
The bark is brewed to make medicinal tea used to reduce fever and as an antiinflammatory drug.
2.3.4. Aromatic:
Roses flowers scent perfumes sachets , potpourris. A recipe used by Scottish chemist
for fifty years. It was purely a liquid one , the essences consisting of Musk , Vanilla ,
Sandal wood, Oatchouli , Verbena , Neroli and Otto of Roses. Roses water is used in
cosmatic industry.
2.3.5. Food :
Rose water may be extracted from rose petals . Rose water may be used to flavor
desserts , pie crusts , chicken dishes and is also used as a wash to protect the skin.
Roses are rich in vitamin C , A and E . Wild rose hips can be eaten . The flowers can
be used for potopourri , eaten raw , made into tea , or jam . Rose petals and fruit are
used as preservatives and rose hips may be used in soups, and as dried fruits . They
are an excellent source of vitamin C (three rose hips contain as much Vitamin C as a
whole orange).
2.3.6. Historical uses:
Romans crowned bridal couples with roses and placed them as banquet centerpieces .
The American Indians used roses medicinally to treat sores , blisters , and as an eye
wash. People have some beliefs about magical properties of roses .Red roses has long
been used for love spells , yellow for luck and rose plants has been grown in garden to
attract fairies who protect their surroundings.
2.3.7. Other uses:
seed are used as decorations , particularly strung together as neck lace. Stems are used
to make baskets. Burnet rose gives a violet dye used in dyeing silk. Silk and moslin
may be dyed with a peach colour taken from the juice of Scotch rose hips .
Anonymous
2.4.
Propagation of Roses:
2.4.1. Traditional techniques:
2.4.1.1.
Propagation by seeds:
Natures own method to renew or increase the rose population is by seed. Seed
propagation is used by rose breeders for the development of new cultivars. Rose seeds
do not germinate readily after harvest. A period of after ripening is necessary before
the seeds are ready to germinate. The fruits or “hips” should be harvested when the
color changes from green to red , yellow or variations of these two colours. Seeds are
removed from the “hips ” sown in flats and stored at a refrigerator 4oC for at least 3-4
weeks. Seed flats are transferred to room a temperature of 18o-21oC and then
germinated . Final germination takes about 2-3 weeks. Seedlings should be
transplanted to a good growing medium for growth untit first bloom.
2.4.1.2.
Cuttings:
Roses should if possible be grown on their own roots, that is from cuttings.
Commercially it is hardly a viable proposition for various reasons: amount of material
required; time taken to produce good saleable plant, particularly Hibrid Teas with
pithy growth , are just not suitable. The percentage of rooted plants is much lower
than that produced by budding. Possible exceptions are some Ramblers and
Miniatures which root readily, but these do not produce better plants than those
budded in the equivalent time. Cuttings can be taken at any time between October and
March depending upon the intended planting date Cutting wood should be selected
from flowering shoots that have been allowed to develop to full bloom. In this way
the propagator is certain that the shoot producing flowers is true to type.
2.4.1.3.
Grafting :
Grafted plants are seldom used for commercial cut flower production. Rosa menetti
under stock is used in the production of grafted plants. It is practiced on the West
Coast of America and Europe. Stocks are field grown for 1 year before being ready to
be grafted .When the under stock are ready for grafting, one eye from the known
cultivar rose shoots should be prepared by making a slanting cut approximately 2-3
cm long at its base. A similar cut is made in the under stock just above the soil line.
“The grafts are tied with a budding rubber”. The graft unions occure in approximately
10 days.Grafted plants shoud be acclimatized for about one month before plantig in
their perinant places.
2.4.1.4.
Budding:
Budded plants are the most popular type used by commercial rose flowers
growers. It has been used for a long time recognized as the commercial method of
propagating roses in all parts of the world. The most common under stock for
budded plants is Rosa manetti with occasional use of Rosa adorata in cold climates.
stock are areas.The budding produced on especially maintained stock blocks normally
segregated from regular growing procedure consists of making a vertical and
horizontal cut in the under stock to from a “T”shaped cut. The “T” is placed well
below the shoots that arise from the under stock An eye is removed Under from a
previously prepared shoot of a named cultivar, inserted into the “T” shaped cut
and wrapped with a budding rubber. Three to 4 weeks after budding , Rosa
manetti under stock is cut back. In warm climates like Sudan Rosa banksiae is
used as an under stock. One of the great advantages of budding is that
only one “eye”is required to produce a plant, a matter of great importance with
new or scarce cultivars. It has to be borne in mind that rose cultivars start off as
one plant; when this is a new and very distinct rose, any early build up of stock is of
considerable significance to the grower. But in this method we need a long time to
produce new plants.
(Larson,1980 ; Gault and synge,1971)
2.4.2 Tissue Culture techniques:
Plant cell and tissue culture techniques have served as methodologies of
physiology and biochemistry in a quest to increase our knowledge of cell biology for
half a century .Today tissue culture has largely been integrated in biotechnology and
permits the regeneration of plants as clones and as transgenics (Vasil and Thorpe
1994) .In vitro culture of higher plants is the culture of embryos ,seeds ,plants ,organs,
tissues , cells or protoplasts on nutrient media under sterile conditions .
(Pierik 1984) .
2.4.2.1.Media used:
Nutrient media require double distilled water .Inorganic and organic
compounds should be research grade .All are soluble in water ,except hormones
which must be dissolved in organic solvents or acid before being added to the medium
.several chemical companies offer formulations of varying degree of completeness so
as to allow for modifications .Catalogues of manufactures of fine chemicals offer
Murashige and Skoog (1962) based salt mixture each without sucrose ,agar ,vitamins
and hormones .they also present a variety of gelling agents ,hormones and other
biochemicals for best choice and preparation .Success in plant cell culture is largely
determined by the quality of nutrient media .Formulations designed by Linsmaier and
Skoog (1965) ,Gamborg ,et al (1972) can be regarded as standard tissue culture media
MS medium was designed to test the effects of organic supplements on growth and
development of tissue cultures . It was standardized with regard to inorganic nutrients
and formulated for tobacco pith tissue culture.It became widely accepted because it
supports the growth and development of cells,tissues,and organs of a wide variety of
plant species ranging from herbaceous species to woody species(astonitiong alike in
size and external appearance the reproducibility was more satisfactory and optimal
yield of new growth were obtained)
(Vasel andThorp,1994).
Plant material and explantation : 2.4.2.2
The scope of plant species employed in tissue culture has been
broadened , particularly due to perceived or actual needs to extend micro propagation
to plants which are of commercial value or are rare and threatened by extinction .
The process of dissection and culture of small organs or tissue sections is
referred to as explanation .Explant choice ,the timing of excision ,and pretreatment
are important determinants of culture success .Healthy ,vigorously growing plants
will render suitable explants .Origin and size of explanted tissue determine the
development of the established culture Number and physiological status of
paranchyma cells subtending the cut surface of the explant will give rise to a
proportional amount of callus . Explanted cells, tissue and organs as well as their
environment must be sterile (Vasil and Thorpe, 1994).
2.4.2.3 Preparation of media:
Nutritional and hormonal requirements of plant tissue and organ
cultures consists of two essential substances: organic compounds like sugars , amino
acids , vitamins ,growth regulators ,undefined mixtures of substances like coco- nut
milk and east extract ; Inorganic compounds (macro and micro mineral elements
like N ,P ,K ,Ca ,Mg , S ,Fe ,Zn ,B ,Mn ,Cu ,Co ,Ni ,Al
,Mo ,I). To determine the right composition of a nutrient
medium for a particular explant it may take at least a year
especially when no information is available on the plant you are
working with .When no agar (a solidifier) is added to a nutrient
medium it is called a liquid medium with agar it is a solid
nutrient medium .There are different ways to express the
concentration of a particular substance the most common terms
are: volume percent ,weight percent ,Molar,Microgram /l g/ml
and PPm .The medium should be sterilized before culturing
.Sterilization is mostly done in autoclaves ,sometimes by way
of filtering and occasionally chemically or by irradiation
.(Pierk 1984).
2.4.2.4 Preparation of explants:
Before an explant is put on a nutrient medium the piece of the plant from which the
explants is cut has to be disinfected .This disinfection is usually done by submerging
the plant piece for a few seconds in 70% alcohol and subsequently in 1.0%-1.5
Na OCL clorex or commercial bleach) for 10-30 minutes .It is then washed (several
times with sterilized water to remove all traces of the bleach,obviously ,disinfection is
an important condition for successful culture in vitro .Nutrient media will fast become
overgrown with molds and bacteria if disinfection has been imperfect .When
sterilizing and preparing tissues it will be necessary to use tweezers ,scalpels , etc
which have been sterilized in (96% alcohol) and flamed before hand
(Pierik 1984)
.
.
.
.
2.4.25 Tissue culture of higher species:
Propagation of herbaceous plants and trees, whether ornamentals, fruit crops
or forest trees by tissue culture techniques has been described as having potential
merit to rapidly increase clones with specific growth characteristics; in large number
necessary for plantation conditions. The application of these techniques to roses may
provide a viable solution to mass production of elite clones with desirable ornamental
traits.
In vitro propagation of woody species has, however lagged behind as
compared to herbaceous species. Until 1977 woody species have been categorized
intractable in culture and thus much less attention has been given to there tissue
culturing. Many reasons have contributed to this state of tissue culture in woody
species. Woody species have long generation time due to a slow growth rate which in
naturally reflected under in vitro conditions where low propagation rates have been
observed (Bonga and Durzan, 1987) besides, growth in woody species occurs in
flushes the number of these flushes depend on environmental conditions. This is
accompanied by the formation of shoots of different ontogenetic age and different
developmental phases greatly influencing the regeneration potential of explants
obtained from them(Murashige, 1977).
Difficulty in disinfestations of explants obtained from woody species has also
played a role in impeding tissue culture of woody species trees and shrubs are usually
grown in open fields under natural conditions for years harbouring various kinds of
contaminants.Another problem associated with woody species tissue culture is the
excessive callus formation at the bases of explant when cultured on nutrient media
(Mogranahan et al.,1987).
Nevertheless, the number of woody plants being propagated successfully by
tissue culture methods has significantly increased in the last decade through out the
world both in terms of crops being propagating or in total number of plants produced
(George and Shrington, 1984, Thorpe et al., 1991 ).
Emphasis is now placed on micropropagation for clonal multiplication of
economically valuable plants, because it can provide greater rates of propagation and
plants that are disease-free. “Sarra” rose cultivar is newly introduced to Sudan.
Mother plants are expensive. Vegetative plant propagules are thus limited. In addition
to that conventional vegetative propagation method have proved difficult, slow,
laborious and are seasonal. Tissue culture techniques have been realized by Jordan
(1987) as highly suitable for the rapid clonal propagation under such cases .
Establishment of an in vitro propagation scheme would enhance cloning of
“Sarra” rose cultivar since propagules for further propagation can be derived from
plantlets growing in vitro circumventing the requirements, for explants from limited
mother plants available. In this way stock-independent in vitro procedure based on recycling of contaminants-free propagules could be be developed on one hand. On the
other hand in vitro propagation of “Sarra” rose cultivar could be carried out all-yearround deturing the seasonal restriction of mother plant growth.
“Sarra” rose cultivar flowers almost around the year affecting greatly the
availability of propagules for conventional propagation methods. Such a problem does
not usually arise and propagation can be done at any time of the year. By the
development of tissue culture propagation system.
Two tissue culture techniques to propagate plants exist: the first of these is the
proliferation and induction of axillary and apical shoot tips. Here shoot apices or
nodal segments each containing at least an axillary bud are excised and cultured on a
nutrient medium. This is the most widely used technique for the clonal propagation of
desired genotype. Plantlet produce are clonal (Murshage, 1974). These plantlets grow
and develop from pre-existing lateral buds. The technique is thus called direct
propagation and the number of plants produced depends on the size of shoot tips
cultured and is genotype-dependent and reflects the spontaneous or induced ability of
geno-type to produce branches. Generally speaking any plant species that produce
branches or respond to that if treated with growth regulators can easily be propagated
by this technique (Murshage 1974, Hussey, 1978). The second technique developed
for in vitro plant propagation involves an initial step of callus formation prior to
induction of adventitious organs (organogenesis) or embryo formation
(embryogenesis). Various reports are available on organogenesis of woody species
(Goyal and Arya, 1981, Barlass and Skene, 1982; Mhatre, et al., 1985; Barbieri and
Marini, 1987; Omura et al., 1987; Amin and Jaiswal, 1087). Embroyogenesis or
asexual embryo formation however has been reviewed by Tisserat, et al., (1979), and
by Durzan (1988).
A wide degree of genetic variability among plantlet produced via callusmediated organ-or embryo-genesis has been observed (Mc Comb and Newton,1981;
Navaro, et al., 1985). The principal of these in vitro propagation technique (called
indirect propagation method ) for plant multiplication has been considered elsewhere
(Evans et al., 1981) ehnce do not warrant elaboration here where a high level of
fidelity in in vitro regenerated plantlets is desired.
The most widely used tissue culture technique in any true-to-type clonal
propagation is the culture of shoot-apices or-segments. (Murashige, 1974; Lawrence,
1981) here both axillary and apical meristems are induced to proliferate and grow into
shoots which can then be rooted and grown into whole plantlet in vitro or ex- vitro or
transferred to fresh medium for further proliferation. The process can be continued
indefinitely provided optimal media and cultural conditions for normal promotion of
vegetative growth in vitro are satisfied. The method of axillary shoot enhancement
has since been applied to a wide range herbaceous and woody species (Murashige,
1974). It has been described as having potential merit to rapidly increase desired
cultivars up to a million-fold in a year (Whitehead and Giles, 1977). It provides
genetic stability as plantlets produced originate directly from performed or newly
formed buds. (D’Amato, 1977).
The procedure of this technique typically involves excision of shoot apices or
stem segments with a single node (nodal cutting), surface disinfestations and culture
in a suitable medium to induce shoot proliferation. Multiple shoots or only a single
shoot may develop depending upon many factors; among which the genotype of plant
under consideration and the culture media are most important. In vitro developed
shoots, in turn, produce axillary and apical buds which can be induced to proliferate
and grow into whole plantlets. Through serial subcultures the process can be repeated
over and over as needed. Nevertheless difficulties still remain concerned vegetative
propagation of woody species by tissue culture methods. These difficulties greatly
affect the rapidity with which plant propagation can be achieved by using the
technique of proliferation of axillary shoots of in vitro cultured shoot apices. The
selection of a suitable source of ex-plant for culture initiation, its conditions, the
juvenile/adult phases, contamination, the slow rate of growth, browning and
vitrification are all but some of the factorsthat are of great importance and can make
the difference between the success and failure of a culture.
Roses have been grown vegitatively by conventional methods such as rooting
of cuttings and grafting. Not more than 10 cuttings generally can be made from a
large rose plant. In addition to seasonality and limited number of mother plants these
methods of propagation has led to disease and nematode infestation during field
culture that can be traced back to the stock material used for propagation. Emphasis is
now placed on micopropagation of economically valuable plants, because it can
provide greater rates of propagation and plants that are disease-free.
There are numerous reports of Rosa sp. tissue cultures including embryo
culture (Lammerts, 1946; Asen and Larsen, 1951; Von Abrams and Hand,1956;
Semeniuk, et al., 1963; Graifenberg, 1973), callus culture (Jacobs, et al., 1968;
Nesius, et al., 1972) and shoot apices culture (Hill, 1976; Jacobs et al., 1969; Jacobs et
al., 1970 a,b; Elliott 1970; Graifenberg et al., 1975; Skirvin and Chu, 1979;
Hasegawa, 1979; Ara et al., 1997; Singh and Syamal, 2000) and protoplast culture
(Kim et al., 2003).
Though Rosa sp. tissue culture has been established in the early fifties no
report on the development of a system for in vitro rose propagation existed until the
late seventies when Skirvin and Chu (1979) achieved shoot proliferation by culturing
shoot tips of “Forever Yours” rose on a modified Murashige and Skoog medium (MS
1962) supplemented with BA (2.0 mg/l) and NAA (0.1 mg/l). shoots were
successfully rooted in vitro on half strength MS medium with out hormones . Rooted
shoots were transferred to green house for acclimatization and they grew well
thereafter . Hasegawa,(1979) also cultured shoot-tips and lateral buds from green
house grown rose plants and obtained multiple shoots with a 6-fold increase in 8
weeks by reculturing in vitro derived shootlets on the the same medium.3.0 mg/liter
BA and 0.3 mg/liter IAA were found to be optimum for the proliferation of multiple
shoots. Rooting was low on a medium containing 0.3 mg /l IAA and devoid of BA.
Rooting of in vitro produced plantlets has been the subject of a number of studies
where the importance of auxin type and amount, salt content of the nutrient medium
or a combination of both influence the rooting process in vitro. Successful
achievement of rooting on media devoid of growth regulators has been reported by
Skirvin and Chu, (1979) and by Hasegawa, (1980).The type of auxin incorporated in
the rooting medium is of vital importance. The auxin IAA and NAA were found to be
effective while IBA was not (Kosh-Khui and Sink,1982), contrary to the finding of
Alderson et al.,(1995) and Ara et al., (1979) where IBA was the rooting hormone of in
vitro produced plantlet.
IAA was reported to induce good rooting but at 10 times the NAA
concentration needed (Hasegawa, 1980). The combination of two auxins were found
to increase rooting more any of the auxins alone (Khosh. Khui and Sink, 1982).
Differences in hormonal requirements for rooting rose cultivars has been reported by
Valles and Boxus (1987). As far as salt concentration is concerned sevrl reports
confirmed the superiority of reduced salt concentration in rooting media for rose
plantlets (Skirvia and Chu, 1979; Davies, 1980; Arnold et al.,1995) and for plants in
general (Murashige, 1979). Low salt concentration in rooting media significantly
increased root initiation for “Improved Blaze” roses (Hyndman et al., 1982) and for
“Rosmanini” dwarf rose cultivar (Scotti-Campos and Pais, 1990). On the other hand,
reduced salt concentration coupled with an auxin (NAA) (Skirvin et al., 1984) or a
combination of auxins (Khosh-Khiu and Sink, 1982) greatly enhanced rooting of “
Improved Blaze” plantlets. The interactive effect of low salt concentrations and auxin
have been extensively studied by Arnold, et al., ()1995 the authers were unsuccessful
in determining the relationship between these two factors.
(Sultanbawa and Phatak, 1991)carried out an experiment
on sterile ornamental peper to propagate it by using two ways, cuttings and in vitro
shoot tip culture .Cuttings were rooted in the green house divided into two treatments
one treated with Rootone and a control treatment.
The second way was by using in vitro shoot tip culture using (MS) medium with 84
mM sucrose ,1% agar, two concentrations of IBA 4.9 or 9.8 µM or 8.8 µM BA in half
strength of MS medium and a control without growth regulators .They found that the
best of cuttings was these treated with Rootone, but when comparing it with in vitro
cultured plants after 8 weeks just 40% of the cuttings have rooted compared with
60%rooted plants after 8 weeks in half strength Ms medium without growth regulators
(Gawel, et al. 1990)worked on in vitro propagation of Miscanthus sinensis
(ornamental plant) by using immature inflorescences of this plant. Three varieties
were cultured on modified MS medium with 9.0 µM 2,4-D,20 g/l sucrose,2.0 g/l Gel
rite, and 0.75 g/l MgCl² .Orango genesis was observed 8-12 weeks after callus
initiation, shoots were rooted on half strength MS medium without growth regulators
.After rooting ,tillers were initiated . Propagation through in vitro tillering is
suggested as a successfull method for propagating this plant .
Auxins , medium salt concentrations , and their interactive effects on rooting
of two winter hardy roses :
( Rosa kordesii Wulf ' John Franklin ' & ' ChamPlain ') and two hybrid teas: ( Rosa
hybrida ' John Paul II ' & ' Landora ' ) were studied .
The auxins ( in mg . liter-1 ) IAA ( 0 , 0.3 , 1.5 , 3.0 , 6.0 , or 15.0 ) , IBA (0 , 0.1 , 0.2
, 0.5 , 1.0 , or 3.0 ) and NAA (0 , 0.1 , 0.2 , 0.5 , 1.0 , or 3.0 ).
Each were combine factorially with modified MS medium ( 1/4 , 1/2 , 1/3 and full
MS concentration. ) and were tested for optimal rooting response . ' ' John Franklin ' , '
John Paul II ' , and ' Landara ' rooted well with low or no auxin medium to high salt
concentration . Optimum rooting for ' Champlain ' was achieved with high IAA and
low salts or with intermediate IBA and NAA concentrations. and low medium salts .
The interactive effects of auxin and medium salts for 'Champlain' showed that as salt
concentration. increased , the amount of IBA or NAA required for optimal rooting
also increased . The effects of auxins and medium concentration. on root counts per
shoot were similar to those for percent rooting . Adding Auxin to the medium reduced
root length for all cultivars , but salt concentration had a minimal effect . Roots
generally were shortest at the highest IBA and NAA conc. salt concentration had little
effect on root length . (Arnold , et al . 1995 ) .
Effect of seven basal media , three carbon sources and four cytokinins on
shoot organogenesis in the chrismas tree Scots pine Pinus sylvestris . was investigated
by ( Ill-Whansul and korban ,1998 )as following :
Seven basal media formulations were selected , they in- cluded MS medium ,
1/2 MS , Gresshoff and Doy (GD) , Schenk and Hild brandt (SH ) , Wolter and Skoog
( WS ) Lloyd and Mc Cown ( Woody plant medium : WPM ) , and Gamborg , et al.
(Gamborg's B5 ) along with three carbon sources including sucrose , glucose and
fructose ( each at a concentration of 58.4 m M ) each medium contained 5 u M BA .
Embryos grown on media lacking any growth regulators were used as control . Effect
of different concentraion of the folowing cytokines BA ,BPA , Zeatin , and TDZ
Provided at four concentrations ( 5 , 10 , 15 , 20 u M ) was investigated .
Hyperhydricity of explants and shoot regenerantes was observed on basal media
containing fructose , especially with half strength MS , MS , and WPM . Explants
grown on a GD medium with sucrose produced the highest frequency of regeneration
(81%) and with no hyperhydricity observed of developing adventitious shoots .
Among three cytokines tested including BA , BPA , and TDZ ( at four concentrations
for each ) . 5 u MBA resulted in the highest regeneration frequency and mean number
of adventitious shoots per embryo . Shoot regenerantes were elongated after transfer
to a GD medium containing 2g-1 activated charcoal and no growth regulators . After
one month rooting was induced on 10% of explants .
Pear Pyrus Communis in vitro propagation experiment using a double phase
culture system was done by ( Rodriguez and Diaz- Sala ,1991 ) using two MS media :
M S 1 ( MS salts plus 100 mg thiamine / Liter ) and M S 2 ( MS salts with half
strength nitrates but double strength calcium chloride and magnesium sulphate plus (
mg / liter ) : 100 myo – inositol , 1 nicotinic acid , 1 pyridoxine . HCL , and ascorbic
acid .
Rooting was accomplished by two methods :
a) Culture on solid MS 2 medium plus IBA ( 30, 10 , 5 µM ) or NAA ( 30, 10 .5
u M) in darkness flowed by 10 to 15 days of culture on a half strength solid
hormone free medium ( MS 2 ) under 16 hr photo period .
Two)1 min immersion in an IBA solution ( 5 , 2.5 , 0.5 , 0.05 µM ) followed by 20
days of culture on a half strength solid hormone free medium ( MS2 ) with a
16 hr photo period . After 40 days there was a clear difference between the
shoot number per explants obtained according to the culture system used .
Shoot multiplication was always higher when a liquid medium was used as an
overlay proliferation of the plant was accomplished with a yield of 10 – 5 new
shoots per explant . Rooting without callus formation was achieved by
immersing the basal end in 5µ M IBA solution for 1 min .
( Balch and Alejo, 1997 ) studied the in vitro plant regeneration of Mexican
Lime and Mandarin by Direct organogenesis starting from seedlings of Mexican
Lime ( Citrus aurantifolia Christem . Swing ) and mandarin ( Citrus reticulate
Blanco cv. Monica ). An other experiment was conducted to investigate the
influence of incubation in several light conditions . The last experiment was to test
the rooting of in vitro generated shoots, adventitious shoots were cultured in a half
strength basal medium amended with 2% sucrose , 0.2% activated charcoal and
supplemented with the following auxins :
2.7 µ M NAA , 5.4 µM NAA , 2.5 µM IBA , and 4.9 µM IBA .
The optimal culture medium from both species was MS medium with
vitamins from B5 medium , 5% sucrose , 33.3 µ M BA and 5.4 µM NAA , The
best response was obtained when the segments were incubated at 25o (+/-) 2oc for
21days in darkness followed by 29 days on a 16/8 h light / dark cycle ( fluorescent
light , 54 µM mol . m-2 s-1 ) . The best regeneration system tested allowed the
attainment of adventitious shoots from 96% and 88% of the explants in Mexican
Lime and Mandarin respectively . In Mexican Lime an average of 7-8 well
differentiated shoots per explants was obtained , and in mandarin the yield was5-1
rooting of 70% . of the shoots was achieved in culture medium with NAA ( 2.7 5.4 u M ) or IBA ( 2.5-4.9 u M ).
An efficient system for in vitro shoot formation and elongation of Dwarf
pomegranate was done by ( Zhang and Stoltz, 1991 ) ; the explants were placed
vertically in culture tubes each containing 10 ml modified MS medium . For shoot
formation and elongation the medium was supplemented with the flowing
combination of plant growth regulators :
With the NAA level fixed at 2.0 µ M ; BA level were (
0.0,0.5,1.0,2.0,4.0,0.8 or 16.0 u M) ; with BA fixed at 2 µ M NAA levels were (
0,0.5,1.0,2.0,4.0,8.0, or 16 µ M ) .The experiment consisted of 14 treatments :
two factors ( BA and NAA ) each with seven levels . Each culture produced a
mean of 5.2 shoots with 1 µ M BA with NAA fixed at 2.0 µ M . BA at other
concentrations resulted in fewer shoots , and at higher levels callus was produced ,
also found BA at 1.0 µ M to result in high shoot production from leaf callus of
tree pomegranate .
There were 6.6 shoots produced per culture with 1.0 µ M NAA with
BA fixed at 2.0 µ M NAA at other concentrations resulted in lower shoot
production , and at higher levels callus was produced . Shoots were > 35 mm long
at BA levels <= 1.0 µ M with NAA fixed at 2.0 µ M . At BA levels >= 2.0 µ M ,
shoots length was< 20 mm . At 8.0 µ M NAA shoot length was 8.0 mm .
Therefore , shoot elongation decreased with BA and NAA conc. > 1.0 and 3.0 µM
respectively. The work showed that auxin NAA at 1.0 µ M is optimal for in vitro
shoot proliferation of dwarf pomegranate and that BA levels > 2.0 µ M inhibit
shoot formation .
A method has been developed for producing putative adventitous
shoots form proliferating shoots of chimeral
Rosa multiflora rootstock . Axillary buds with an internodal section ( 1 cm ) were
explanted onto the modified MS medium developed for roses ( Skirvin and Chu
1979) containing BA 2.0 mg-1 . When single shoot micro cuttings were
subcultured onto the same medium at 3-4 week intervals the shoots fail to
elongate more than 1-1.5 cm , new leaflets were small and the basal levels began
to senesce . Because the leaf senesce observed in the cultures resembled classic
ethylene injury . The problem might be overcome by reducing ethylene levels in
vitro by use of an inhibitor of ethylene synthesis like silver nitrate . shoot
proliferation was studied by studying the effect of TDZ at three concentrations (
0.5 , 1.0 , and 1.5 µ M ) alone or in combination with three levels of NAA ( 0.0,
0.05,and 1.0 mg/L ) . Rooting was studied after harvesting shoots ( 2-3cm tall )
from proliferating cultures ; cultures were moved to several rooting media that
contained various auxins , sugars , as well as activated charcoal , silver nitrate and
GA3 . There were 6 media M ( MS 0.0 , growth regulators 0.0 , fructose 20 ,
sucrose 20 ) , CH ( 1/3 MS , NAA 0.1 , sucrose 30 ) , R5 ( ½ MS , NAA 0.1 ,
IAA 0.5 , glucose 20 , fructose 20 ) , R9 ( MS , GA3 0.1 , NAA 0.05 , IAA 0.5 ,
IBA 0.05 , glucose 20 , fructose 20 ) , R10 ( MS , GA3 0.5 , NAA 0.5 , IAA 1.0 ,
IBA 0.5 , AgNO3 3.4 , sucrose 40 , active charcoal 0.2 gm/1 ) , all sugars in g/1
and all growth regulators in mg/1 . The best rooting ( 50% was observed on R10
medium which produced healthy shoots and 50% rooting . Subculture on medium
with 1µM TDZ development compact nodular callus that later after one or two
subcultures onto the same medium , formed putative adventitious shoots . About
half of these shoots rooted on MS medium supplemented with three auxins ( NAA
0.5 mg L/1 , IAA 1.0 mg /1 , IBA 0.5 mg /1 , GA3 0.5 mg /1 , silver nitrate 3.4 mg
/1 , activated charcoal 200 mg /1 , and sucrose 40 g /1 ) ( Rosu , et al. , 1995 ) .
( Nayak , et al., 1997 ) studied in vitro propagation of three epiphytic
orchids through TDZ to induce high frequency
shoot proliferation ; The medium of MS containing 100 mg /1 ( w/v ) myoinositol and 3% (w/v ) sucrose was used . This medium was further supplemented
whith 4.4 - 44 µ M BA or 0.045 – 9.0 µ M TDZ , either individually or in
combination with 5.4 – 27 µ M NAA , with 0.8% (w/v) agar . For rooting of
shoots MS medium was used with 0.2% phytagel and 3% sucrose . The medium
was further supplemented with IBA or NAA 5.4 – 10.8 µ M . MS medium
containing BA or TDZ with the latter begin more effective at 2.2 – 4.5 µ M .
Shoots which developed on a TDZ containing medium elongated following
transfer to a medium containing 2.2 µ M BA and 10.8 µ M NAA conc . of TDZ
above the optimal level had an inhibitory effect on shoot regeneration . In both
Dendrobium species the number of shoot bud formation was greatly influenced by
explant orientation . Regenerated s hoots were rooted on MS containing 10.8 µ M
IBA .
A study was conducted to develop a thornless Black berry ( Rubus spp
) cultivar called Navaho , The initial medium was one half strength MS with 30 g
sucrose and 0.3 g acid washed activated charcoal/l . Nodal segments were then
transferred to jars for 3 week of proliferation . The jars contained 2.5 ml of one of
the media : full strength MS plus 0.3 g acid washed activated charcoal /l (A) ; full
strength MS ( B) and one half strength MS(C) . All three media were
supplemented with 30.0 g sucrose / l , 7.0 g agar / liter , 8.9 µ M BA , 0.5 µ M
IBA , 0.29 µ M GA . Nodal segments cultured in medium (A) produced vigorous
non proliferating shoots with several long roots . Nodal segments cultured on (B)
and(C )were very similar and formed three to five adventitious shoots from (B)
and (C) were divided and subcultured on the same respective media for continued
proliferation , although micro shoots on (B) developed slight chlorosis . Single
micro shoots , removed from proliferating clumps from (B) and (C) and
transferred to (A) grew vigorous with numeric roots with out shoot proliferation .
About 90% of all micro shoots transferred to a produced roots with in 2 week ,
with the only losses due to contamination of the vessel ( Fernandes and Clark
1991 ) .
Easter lily ( Lilium longiflorum Tunb.) was in vitro propagated from
pedicels by ( Liu and Burger ,1986) ,pedicel sections from the flowers were
cultured on a modified MS medium containing various concentration of
cytokinins and auxins , two cytokinins kinetin and BAP and two auxins IAA and
NAA were used in several combinations . These growth regulators were added to
a basal medium consisting of MS salts, 87.6mM (3%) sucrose , 555 µ M (100 mg
/1) myo-inositol . 1.2 u M(0.4 mg /1) thiamin and HCL , and 0.6% phytagar . The
medium tested for root initiation consisted of an MS medium containing no auxin
( control ) or 10 µ M of either IAA , IBA , or NAA . A fifthmedium contained 20
µ M NAA . A combination of BAP (5 µ M ) and NAA ( 2 µ M ) resulted in the
greatest number of adventitious buds on pedicel section . A gradient in the
formation of buds in the pedicel was observed , with the section nearest the
receptacle forming the greatest number , especially when the section was placed
upside-down on the culture medium . IAA ( 10 µ M ) and IBA ( 10 µ M ) were
most effective in stimulating adventitious roots in vitro derived shoots . This
vegetative propagation technique provides a way of amplifying floral mutants of
Easter lily .
Cotyledons from developing 6-8 week old embryos of Liatris Spicata (L) Wild . (
blazing star ) were cultured on MS medium containing 0.0 , 0.4 ,4.4 , or 44.4 µ M
BA or 0.0 , 0.2 , 2.2 , or 22.2 µ M TDZ to induce adventitious shoot formation .
the highest percentage of cotyledons forming the most shoots was on medium
containing 2.2 µ M TDZ . Cotyledon derived callus cultured on medium
containing 4.4 µM BA formed = 16 times more adventitious shoots than on 2.2 µ
M TDZ . Adventitious shoots derived from cotyledons or callus produced roots
when placed on MS medium containing 5.0 µ M IBA . Regenerated plants that
flowered in the field appeared homogeneous. ( Stim- art and Mather ,1996 ) .
Four muscandine grape ( Vitus rotundifolia Michx ) cultivars ( Carlos ,
Noble , Regale , and Tarheel ) were evaluated for their ability to be cultured in
vitro . Axillary buds were placed on MS medium as modified by Chee . Different
levels of BA 0.5 to 10.0 µ M , Kinetin 0.5 to 5.0 µ M , and TDZ 0.5 to 11.3 µ M ,
and different explant position were evaluated for their effect on in vitro explant
establishment and shoot production . TDZ ( 2.3 to 4.5 µ M ) alone or in
combination with BA ( 1.0 to 5.0 µ M ) or kin ( 1.0 or 5.0 µ M ) was defective for
establishing axillary buds . Similar levels were also effective for promoting shoot
proliferation . Explants originating from the 10 basal nodes of a shoot with at least
25 nodes gave better shoot proliferation than explants originating from the 10
distal nodes . ( Sudarsono and Goldy ,1991 ) .
Cotton fibers were tested as a substitute for agar in tissue culture by (
Moraes – Cerdeira ,1995 ) . The cost of agar has prompted the researcher to search
for an alternative more economical medium support . Effectiveness as a medium
support was evaluated in terms of callus maintenance and shoot organogenesis
using Artemisia , Agrostis and Taxus plants . Taxus and Agrostis calli cultivated
on liquid media with cotton fiber as medium support ( 25 mL of medium per gram
of cotton ) grew better than calli on agar ( 0.8% w/v ) . There were no significant
differences in shoot organogenesis of Artimisia and Agrostis grown in 25 ml of
medium per gram of cotton from those grown in agar medium .
A modified culture medium is presented that promotes in vitro rooting
of grapevine rootstocks and (Vitis vinifera L.)growth regulators .Study of 15
Vitis genotypes indicated a strong genotype dependent response to culture
medium and growth regulators with respect to formation of roots in one node
shoot segments .The media used were MS ,PH 5.7 ,and a medium which they
had designated Roubelakis ,Containing (in mg.l ) NH4NO3 500,KNO3 1000
,CaCl2.2H2O
200,
MgSO4.7H2O1.0
,KI
0.5
,CuSO4.5H2O
0.01,CoCl2.6H2O 0.01 ,ETDA 40 ,biotin 0.1 ,nicotinic acid 5, pyridoxine
5,thiamin 5,panthothenic acid 5 ,myo- inositol 100 ,Sucrose 2%(W/V) and
agar 0.7% (W/V) pH 6.4 .IBA was added at 1,2,3,5,8, µM .The affect of the
antioxidant citric acid at µM
And of activated charcoal at 3% (W/V) were also tested . In preliminary experiments
on rhzoegenesis ,IBA gave superior results to IAA,NAA ,2,4-D and was further used
in this work .The number of roots forming on one node segments in the rootstock and
Vitis vinifera cultivars was determined .Of the 15 genotypes that were studied seven
did not form roots in MS medium with out IBA ,whereas the remaining eight
developed poor root systems .In Roublelakis medium without IBA ,almost all
genotypes developed strong root systems ,with the exception of (Liatiko) and
(SO4)which perform poorly in both media in the absence (Liatiko)
or in the
presence (SO4) of IBA.
(Roubelakis – Angelakis and Zivanovitc ,1991)
3- Materials & Methods
3.1 Plant Material:
The plant material for this study was selected from vigorously growing one
year old plants of roses “Sarra cultivar”. Branches 1.5cm in length containing axillary
buds were taken from these plants for explant prepration.
3.2 Sterilization:
The branches were first washed under running tap water to remove dust and
reduce the contamination. The axillary buds were placed in an antioxidant solution
(100 mg/l citric acid + 150 mg/l ascorbic acid) for one hour. They were then
disinfested under a laminar-air-flow cabinet by dipping in 75% ethanol (V/V) for few
seconds, rinsed by sterilized distilled water followed by immersion in 10%
commercial bleach (Clorox) containing two drops of Tween-20 per 100 ml as a
wetting agent shaken for 15 minutes on a shaker. The axillary buds were then rinsed
three times with sterilized distilled water to remove all of the sterilant.
3.3 Culture media:
The salt formulation used through out this study was that of Murshage and
Skoog, (1962). The components of each stock solution this salt mix is given in
appendix (1).
In the beginning three basal media were tested for there efficiency in
supporting the growth and the development of rose explants. These three media are
Murashuge Multiplication Medium A (MMMA), Murashuge Multiplication Medium
B (MMMB), and Murashuge Multiplication Medium C (MMMC). The chemical
composition of each of these media is shown in appendix (2). Medium preparation
and sterilization were carried out as usual. A one liter medium was prepared of each
three media. The chemical components of each medium was mixed and the pH was
adjusted to 5.7±1 with 0.1 N(HCL) or 0.1N (NaOH) before the addition of agar. The
medium was then heated to dissolve the agar and then distributed into 15 × 150ml
culture tubes at a rate 25ml each tube. The tubes were closed with Bellco Kaputs
closures. The tubes containing the medium were sterilized at 121˚C for 15 minute at
15 psi in an outoclave. The medium was cooled slanted and stored until use.
3.4 Culture of explant:
Sterile axillary buds were established, one axillary bud per test tube a total of
10 axillary buds for each of three Murshage multiplication media. Cultured tubes
were incubated at 20±/1˚C and under 8 hour photoperiod in the growth room. Data
were recorded after 6 weeks incubation period. Data recorded included plant height,
number of shoots, number of leaves, and number of roots produced. Growth vigor was
also recorded visually.
The result of this initial experiment showed that Murashuge Multiplication
Medium (A) (MMMA)was superior to the other two multiplication media in all
parameters measured (plate 1). It was thus decided to use Murashige multiplication
medium (A) (MMMA) as basal medium through out this study.
3.5 Experimentations:
3.5.1 The effect of different concentrations of Murashige and Skoog (1962) salt
mixture on in vitro growth and development of rose axillary buds:
MS salt concentrations were tested as follows:
0.25 x, 0.5 x, 1.0 x (full MS salt concentration), 2.0 x, and 4.0 x.
Plate (1)
3.5.2 The effect of different concentrations of NaH2PO4.H2O salt on
in vitro growth and development of rose axillary buds:
The following concentrations of sodium hypophosphate were tested:
0.25 x, 0.5 x, 1.0 x, 2.0 x, and 4.0 x.
3.5.3 Energy source (Carbohydrates):
3.5.3.1 Sucrose:
It’s the most used of the carbohydrates as an energy source in plant tissue
culture media.
It was tested at: 0.75%, 1.5%, 3.0%, 6.0% and12%.
3.5.3.2 Glucose:
It’s a monosaccharide has been found to be superior or equal to sucrose as an
essential medium component especially for in vitro culture of monocots. A
concentration test of glucose was conducted using the following concentrations:
0.75%, 1.5%, 3.0%, 6.0%, and 12.0%.
3.5.4 the effect of different concentrations of Thiamine – HCL (Vit. B1) on in vitro
culture rose axillary buds:
Thiamine –HCL is one of the (B) complex group of vitamins has been sited as
the most important vitamin in plant tissue culture the essentiality of which has been
proven. A test has been conduced to determine the best concentration for sore axillary
buds growth and development. The following concentrations (in mg/l) has been
tested: 0.1, 0.2, 0.4, 0.8, and 1.6.
3.5.5 The effect of different concentrations of myo-inositol on in vitro cultured
rose axillary buds:
the hexitol myo-inositol is a pentose suger. It has been used in tissue culture
medium as a vitamin. The following concentrations (in mg/l) have been tested:
25,50,100,200, and 400.
3.5.6 The effect of different concentrations of adenine sulphate (A/S) on in vitro
cultured rose axillary buds:
Concentrations of A/S tested included (in mg/l):
20, 40, 80, 160, and 320.
3.5.7 The effect of different concentrations of Naphthaline acetic acid (NAA) and
benzyle adenine (BA) alone and in combination on in vitro growth of rose
axillary buds:
A factorial experiment was conducted to determine the optimum
concentration(s) most suitable for rose axillary buds growth are proliferation.
The following concentrations of (NAA) (in mg/l) were tested: 0.0, 0.01, 0.03,
0.1, and 0.3.
The concentrations of BA tested in (in mg/l) were:
0.0, 0.1, 0.3, 1.0, and 3.0.
3.5.8The effect of physical state of the nutrient medium on the growth and
development of in vitro cultured rose axillary buds:
A comparison of the growth rose of explants on a agar solidified nutrient medium
(7.000 mg/l) with there growth on a plate form made of cotton fibber was conducted.
3.5.9 The effect of darkness on growth and development of in vitro cultured rose
axillary buds:
The experimental layout was as follows:
Cultured plantlets were incubated under the normal incubation
photoperiod (8 hours dark/16 hours light) as control. The dark treatments consist of
growing cultured plantlets under continuous darkness before being exposed to the
normal incubation photoperiod. Cultured planets were incubated under continuous
darkness for one week; two weeks; three weeks and four weeks before being exposed
to normal incubation photoperiod.
The dark condition under incubation was imposed by surrounding a shelf in
the incubation room with black cloths.
3.6 Statistical analysis:
The experimental layout was a completely randomized design. Data were
recorded every two weeks for a total of six weeks for each test. Parameters measured
included:
Plant height, number of leaves, number of nodes, number of shoots, number of
roots if any, callus formation, and overall plant vigar.
A computer program (SAS) was use in the analysis of data.
4 - Results
4.1 Effect of different concentrations of Murashige and Skoog (MS
1962) salt mix on growth and development of rose axillary buds:
Table (1) shows that there were significant differences between the five
concentrations of MS medium salt mix tested in all parameters. The concentration 1.0
x was the best of all in plant height, number of leaves (Fig.1) number of nodes, and
number of shoots. Very high or low concentrations (4.0x, 0.25x) resulted in adverse
effects in all growth parameter measured (Plate 2).
4.2 Effect of different concentrations of NaH2PO4 salt on growth and development of rose axillary
buds:
Generally there was significant reduction in growth and development when
using 0.25x and 4.0x after two and four weeks from culture but differences became
more significant after 6 weeks form culture for all parameters recorded. Table (2)
indicates that the highest growth rates in plant height (Fig. 2), number of leaves
(Fig.3), number of nodes, and number of shoots were obtained at 1.0x concentration,
the normal concentration usually used in tissue culture media.
4.3 Effect of energy sources (carbohydrates):
4.3.1 Effect of different concentrations of sucrose on growth and development
of rose axillary buds:
Table (3) shows that increase of sucrose concentration from 0.75% to 3.0%
resulted in significant increase in all parameters measured, namely plant height (Fig.
4), number of leaves (Fig. 5), number of nodes, and number of shoots per plant. The
concentration 3.0% was the best one while lower and higher concentrations
significantly reduced growth rates (Plate 3).
4.3.2 Effect of different concentrations of glucose on growth and development of
rose axillary buds:
Table (4) shows that all parameters measured increased with increasing glucose
concentraton until the concentration of 3.0% where best results were reached. This
increase was not significant in the first two weeks but significant reduction was
obtained at 12.0%. as for weeks 4 and 6 there is generally significant increase up to
3.0% followed
by decrease at 12.0% concentration. Fig. (6) and (7) show effect of different glucose
concentrations on number of shoots and nodes. Plate (4) shows rose plantlets growing
on different glucose concentrations.
Table 1:Effect of different concentrations of MS salts on in vitro growth
and
development of rose axillary buds.
Weeks
After
culture
trea t.
A
B
plant
hieght
(cm)
1.3 b
1.24 bc
No.of
leaves
5.8 c
6.8 c
No.of
nodes
No.of
shoots
No.of
roots
Root
length
(cm)
5.8 c
1.4 bc
0.0
0.0
6.8 c
1c
0.0
0.0
0.0
0.0
C
1.68 a 10.8 a
10.8 a 2.4 a
D
1.34 b 8.4 b
8.4 b
1.8 ab 0.0
0.0
E
1.1 c
3.2 d
1c
0.0
0.0
A
1.28 c 7.6 bc
7.6bc
1.4 c
0.0
0.0
1.4 c
9.2 b
9.2 b
1.2 c
0.0
0.0
C
2.4 a
19.2 a
19.2 a 3.2 a
0.0
0.0
D
1.82 b 16.4 a
16.4 a 2.2 b
0.0
0.0
B
3.2d
E
1.16 c 4.8 c
4.8 c
1c
0.0
0.0
A
1.24 d 8.4 b
8.4 b
1.2 c
0.0
0.0
1.66 c
9.6 b
9.6 b
1c
0.0
0.0
2.96 a
23.2 a
23.2 a
3.6 a
0.0
0.0
2.46 b
20 a
20 a
2.8 b
0.0
0.0
1.18 d
4.6 c
4.6 c
1c
0.0
0.0
B
C
D
E
A= 0.25x B = 0.50x C = 1.0x D = 2.0x E = 4.0x
Means with the same letter(s) are not significantly different using
Duncan Multiple Range Test .
Fig. 1: Effect of MS salts mix concentrationon number of leaves
of in vitro cultured plantlets of "Sarra" rose cultivar.
A = 0.25X
B = 0.50X
C = 1.00X
D = 2.00X
E = 4.00X
Plate 2
Table 2:Effect of different concentrations of Na H2 PO4
salt on in
vitro
growth and development of rose axillary buds.
Weeks
After
culture
plant
trea t.
A
B
C
No.of
leaves
hieght
(cm)
1.22 b
1.58 a
8c
12.6 ab
1.54 a 12.6 ab
D
1.6 a
E
1.26 b 9.2 bc
A
B
14.0 a
No.of
nodes
No.of
shoots
no.of
roots
Root
length
(cm)
8c
1.6 ab
0.0
0.0
12.6 ab
2.2 a
0.0
0.0
12.6
2.2 a
ab
14.0 a 2.0 ab
0.0
0.0
0.0
0.0
9.2 bc 1.2 b
0.0
0.0
1.26 b 10.4 b
10.4 b 1.4 b
0.0
0.0
1.64 a
14.6 a
0.0
0.0
14.6 a
2 ab
C
1.72 a 16.8 a
16.8 a 2.8 a
0.0
0.0
D
1.68 a 17 a
17 a
0.0
0.0
E
1.24 b 14.4 b
10.4 b 1.4 b
0.0
0.0
A
1.3 c
11.4 b
11.4 b 1.8 b
0.0
0.0
2.06 b
19.2 a
19.2 a
2.8 ab
0.0
0.0
2.58 a
21 a
21. a
3.6 a
0.0
0.0
B
C
2.2 ab
D
E
2.28 ab
20.6 a
20.6 a
2.8 ab
0.0
0.0
1.3 c
12.6 b
12.6 b
1.4 b
0.0
0.0
A= 0.25x B = 0.50x C = 1.0x D = 2.0x E = 4.0x
Means with the same letter(s) are not significantly different using
Duncan Multiple Range Test.
Fig. 2: Effect of different concentrations of NaH2PO4 salt on
plant height (cm)of in vitro cultured plantlets "Sarra"cultivar
A = 0.25X
B = 0.50X
C = 1.00X
D = 2.00X
E = 4.00X
Fig. 3: Effect of different concentrations of NaH2PO4 salt on number of leaves
of in vitro cultured plantlets of "Sarra"rose cultivar
A = 0.25X
B = 0.50X
C = 1.00X
D = 2.00X
E = 4.00X
Table 3:Effect of different concentrations of sucrose on in vitro growth
and
development of rose axillary buds.
Weeks
After
culture
plant
trea t.
hieght
(cm)
1.34 bc
No.of
leaves
No.of
nodes
No.of
shoots
No.of
roots
Root
length
(cm)
6 bc
1b
0.0
0.0
8.6 ab
1.4 ab
0.0
0.0
10.4 a
2a
0.0
0.0
D
1.52
10.4 a
ab
1.22 c 10.2 a
10.2 a
1.8 a
0.0
0.0
E
1.08 c 5.4 c
5.4 c
1b
0.0
0.0
A
1.3 b
15.4 c
15.4 c
2.8 ab
0.0
0.0
1.8 a
20.6 ab
20.6 ab
2.6 ab
0.0
0.0
C
1.9 a
21.4 a
21.4 a
3.8 a
0.0
0.0
D
1.2 b
16.4 bc 16.4 bc 2.2 b
0.0
0.0
E
1.02 b 6.8 b
6.8 d
1c
0.0
0.0
A
1.44 c 23.4 b
23.4 b
3.2 bc
0.0
0.0
1.74 b
25.6 ab
25.6 ab
4.2 a
0.0
0.0
2.6 a
28 a
28 a
3.8 ab
0.0
0.0
1.3 cd
23.4 b
23.4 b
2.8 c
0.0
0.0
1.06 d
7.4 c
7.4 c
1.2 d
0.0
0.0
A
1.64 a
6 bc
8.6 ab
B
C
B
B
C
D
E
A= 0.75%
B = 1.5% C = 3.0%
D =6.0% E =12.0%
Means with the same letters are not significantly different using
Duncan Multiple Range Test .
Fig. 4: Effect of different concentrations of sucrose on plant height (cm) of in vitro
cultured plantlets of "Sara" rose cultivar.
A = 0.75%
B =1.5%
C = 3.0%
D = 6.0%
E = 12.0%
Fig. 5: Effect of different concentration of sucrose on number of leaves of
in vitro cultured plantlets of"Sarra" rose cultivar
A = 0.75%
B =1.5%
C = 3.0%
D = 6.0%
E = 12.0%
Plate (3)
Table 4:Effects of different concentrations of glucose on in viro growth
and
development of rose axillary buds.
Weeks
After
culture
plant
trea t.
A
B
hieght
(cm)
1.36 a
1.28 a
No.of
leaves
7.6 a
9.8 a
No.of
nods
No.of
shoots
No.of
roots
Root
length
(cm)
7.6 a
1.2 b
0.0
0.0
9.8 a
2.6 a
0.0
0.0
C
1.44 a 9.8 a
9.8 a
2.6 a
0.0
0.0
D
1.24 a 9.0a
9.0 a
3.2 a
0.0
0.0
E
1.18 a 4.2 b
4.2 b
1.2 b
0.0
0.0
A
1.8 b
13 c
13 c
2 bc
0.0
0.0
1.78b
14.6 bc
14.6 bc
2.8 ab
0.0
0.0
18.8 a
3.2 a
0.0
0.0
17.4 ab 17.4 ab 3.4 a
0.0
0.0
B
C
2.16 a 18.8 a
D
1.7 b
E
1.14 c 5.4 d
1.2 c
0.0
0.0
A
1.14 c 13.54 c 13.54 c 2.4 b
0.0
0.0
2.22 c
22 b
22 b
3.8 a
0.0
0.0
3.72 a
30.2 a
30.2 a
4.4 a
0.0
0.0
2.86 b
29 a
29 a
4.6 a
0.0
0.0
1.20 d
6.4 d
6.4 d
1.4 c
0.0
0.0
B
C
D
E
5.4 d
A= 0.75% B = 1.5 %C = 3.0% D = 6.0% E = 12.0%
Means with the same letter(s) are not significantly different using
Duncan Multiple Range Test .
Fig. 6: Effect of different concentrations of glucose on number of shoots of
in vitro cultured plantlets of "Sarra"rose cultivar
A = 0.75%
B =1.5%
C = 3.0%
D = 6.0%
E = 12.0%
Fig. 7: Effect of different concentrations of glucose on number of nodes of
in vitro cultured plantlets of "Sarra" rose cultivar
A = 0.75%
B =1.5%
C = 3.0%
D = 6.0%
E = 12.0%
Plate (4)
4.4 Effect of different concentrations of Thiamine HCL (Vitamin B1)
on growth and development of rose axillary buds:
Table (5) shows that there was a significant increase in all parameters
measured with an increase in thiamin-HCL concentration until 0.8 mg/l was reached
which gave the best results in plant height (Fig. 8) and number of shoots (Fig. 9) after
6 weeks from cultures while the concentration 0.4mg/l was the best on the number of
leaves and nodes. Growth rate decrease at 1.6mg/l plate (5) shows rose plantlets
growing on different thiamine-HCL concentrations.
4.5 Effect of different concentrations of Adenine Sulphate (A/S) on growth and
development of rose axillary buds:
Table (6) shows that there was a little difference on growth rates: (Plant
height, number of leaves (Fig. 10), number of nodes, and number of shoots (Fig. 11)
between the five concentrations tested. Plate (6) shows rose plantlets growing on
different A/S concentrations.
4.6 Effect of different concentrations of myo-inositol on growth and development
of rose axillary buds:
Table (7) shows that increasing the concentration of myo-inositol has no effect
on plant growth rates after six weeks from culture where no significant differences
between the different concentrations was observed, with only slight difference after
two or four weeks on number of leaves (Fig. 12) and nodes but there were no
significant differences on plant height (Fig. 13) and number of shoots.
4.7 Effect of different concentrations of growth regulators:
4.7.1 Naphthaline acetic acid (NAA):
Table (8) shows that changes of NAA concentrations has no effect on plant
height, number of leaves, number of nodes, and number of shoots after two or four
weeks form culture but after six weeks from cluture higher concentrations
significantly increased plant height (Fig. 14), number of roots (Fig. 15) and root
length. Number of roots was also increased after four weeks from culture. Plate (7)
shows rose plantlets growing on different NAA concentrations.
4.7.2 Benzyle adenine (BA):
Table (9) shows that there was significant increase in plant height (Fig.
16),number of leaves, number of nodes, and number of shoots (Fig. 17) with increase
in BA concentrations. The concentration (0.3mg/l) was the best for all parameters
measured except number of shoots where (1.0mg/l) was the best. Increasing the
concentrations to
3.0mg/l growth rates of all parameters measured decreased. Roots were induced on
plantlets grown on medium free of growth regulators and on lowest concentrations
(0.1mg/l). plate (8) shows rose plantlets growing on different BA concentrations.
Table 5:Effect of different concentrations of thiamine – HCl on in
vitro
growth and development of rose axillary buds.
Weeks
After
culture
plant
trea t.
mg/l
A
B
C
D
hight
(cm)
1.16 b
1.3 ab
No.of
leaves
7.4bc
8.8 b
No.of
nodes
No.of
shoots
No.of
roots
Root
length
(cm)
7.4 bc
1.16 b
0.0
0.0
8.8 b
2 ab
0.0
0.0
1.36 b 20.2 20.2 3.2 a 0.0
a
a
1.44 a 9.2 b 9.2 b 2 ab 0.0
0.0
0.0
E
A
B
C
D
E
A
B
C
D
E
1.28
5.6 c 5.6 c 1.6
0.0
ab
ab
1.24 b 11.8 11.8 2.0 a 0.0
b
b
0.0
1.42 b
0.0
14 b
0.0
0.0
14 b
2.2 a
1.36 b 20.2
a
2.2 a 19.4
a
1.32 b 12.2
b
1.3 b 14.8
b
20.2
a
19.4
a
12.2
b
14.8
b
3.2 a 0.0
0.0
2.4 a 0.0
0.0
2.2 a 0.0
0.0
2.2 c 0.0
0.0
1.46 b
19 b
19 b
0.0
0.0
1.6 b
27.0 a
27.0a
2.6
abc
3.6 a
0.0
0.0
2.36 a
25.8 a
25.8 a
3.8 a
0.0
0.0
1.32 b
15.4 b
15.4 b
2.4 bc
0.0
0.0
A= 0.1 B = 0.2 C = 0.4 D = 0.8 E = 1.6 all in mg /l
Means with the same letter(s) are not significantly different using
Duncan Multiple Range Test .
Fig. 8: Effect of different concentrations of Thiamin-HCl on plant height (cm) of
in vitro cultured plantlets of "Sarra"rose cultivar
A =0.1
B =0.2
C =0.4
D = 0.8
E = 1.6 all in mg/l
Fig. 9: Effect of different concentrations of Thiamin-HCl on number of shoots of
in vitro cultured plantlets of "Sarra"rose cultivar
A =0.1
B =0.2
C =0.4
D = 0.8
E = 1.6 all in mg/l
Plate (5)
Table 6:Effect of different concentrations of adenine sulphate on in
vitro
Weeks
After
culture
growth and development of rose axillary buds .
Treat
A
Plant
Hieght
(cm)
1.14 a
No.of leaves
6.8 ab
No.of
nodes
No.of shoots
No.of roots
Root
length
(cm)
6.8 ab
1a
0.0
0.0
1.2 a
0.0
0.0
1.24 a
7.6 a
7.6 a
C
1.16 a
6.6 ab
6.6 ab 1 a
0.0
0.0
D
1.14 a
8a
8a
1a
0.0
0.0
E
1.14 a
6b
6b
1a
0.0
0.0
A
1.36 a
20.4 a
20.4 a 3.6 a
0.0
0.0
1.76 a
19.4 a
19.4 a
2.6 ab
0.0
0.0
1.46 a
17 ab
17 ab
2.4 b
0.0
0.0
1.44 a
17.6 ab 17.6
ab
2.8 a
0.0
0.0
B
B
C
D
E
1.52 a
14.2 b
14.2 b 2.8 a
0.0
0.0
A
1.6 ab
26.4 a
26.4 a 4.2 a
0.0
0.0
1.86 a
25.2 a
25.2 a
3.6 b
0.0
0.0
1.58 b
23 ab
23 ab
3.2 b
0.0
0.0
1.54 b
22.8 ab
22.8 ab
3.4 b
0.0
0.0
1.52 b
19.8 b
19.8 b
3.2 b
0.0
0.0
B
C
D
E
A =20 B =40 C = 80 D = 160 E = 320 ( all in mg /l )
Means with the same letter(s) are not significantly different using
Duncan Multiple Range Test .
Fig. 10: Effect of different concentrations of Adenine sulphate on number of
of in vitro cultured plantlets of "Sarra"rose cultivar
leaves
A =20
B =40
C =80
D =160
E =320 all in mg/l
Fig. 11: Effect of different cocentrations of adenine sulphate on number of
shoots of in vitro cultured plantlets of"Sarra" rose cultivar
A =20
B =40
C =80
D =160
E =320 all in mg/l
Plate(6)
Table7:Effect of different concentrations of myo –inositol on in vitro
growth
and development of rose axillary buds.
plant
Weeks
After
culture
trea t.
A
B
hight
(cm)
1.08 a
1.12 a
No.of
leaves
5.4 a
3.4 b
No.of
nodes
No.of
shoots
No.of
roots
Root
length
(cm)
5.4 a
1a
0.0
0.0
3.4 b
1.0 a
0.0
0.0
C
1.12 a 4.8 ab 4.8 ab 1.0 a
0.0
0.0
D
1.16 a 5.8 a
1.0 a
0.0
0.0
E
1.14 a 4.8 ab 4.8 ab 1.0a
0.0
0.0
A
1.26 a 19.4 a 19.4 a 3 .0a
0.0
0.0
1.26 a
0.0
0.0
1.34 a 17.8 a 17.8 a 2.8 a
0.0
0.0
3.0a
0.0
0.0
E
1.34 a 17.4
ab
1.34 a 14 b
2.8 a
0.0
0.0
A
1.44 a 24.4 a 24.4 a 3.2 a
0.0
0.0
1.54 a
25.2 a
25.2 a
3.8 a
0.0
0.0
1.5 a
24.2 a
24.2 a
3.6 a
0.0
0.0
1.44 a
23.6 a
23.6 a
4.4 a
0.0
0.0
B
C
D
15.4 b
5.8 a
15.4 b
17.4
ab
14 b
2.8 a
B
C
D
E
1.5 a
22.6 a
22.6 a
3.6 a
0.0
0.0
A= 25 B = 50 C = 100 D = 200 E = 400 ( all in mg /l)
Means with the same letter(s)are not significantly different using
Duncan Multiple Range Test .
Fig. 12: Effect of different concentrations of myo-inositol on number of leaves
of in vitro cultured plantlets of "Sarra" rose cultivar
A =25
B =50
C =100
D =200
E = 400 all in mg/l
Fig. 13: Effect of different concentrations of myo-inositol on plant height (cm) of
in vitro cultured plantlets of "Sarra" rose cultivar
A =25
B =50
C =100
D =200
E = 400 all in mg/l
Table 8:Effect of different concentrations of NAA on in vitro growth
and
development of rose axillary buds.
Weeks
After
culture
plant
trea t.
0.0
0.01
hight
(cm)
1.9 a
1.7 a
No.of
leaves
5.4 a
3.6 a
No.of
nodes
No.of
shoots
No.of
roots
Root
length
(cm)
5.4 a
1a
0.0
0.0
3.6 a
1a
0.0
0.0
0.03
1.84 a 4.6 a
4.6 a
1.4 a
0.0
0.0
0.1
1.94 a 3.6 a
3.6 a
1a
0.0
0.0
0.3
1.96 a 4 a
4a
1a
0.0
0.0
2.2 a
6.4 a
6.4 a
1.2 a
1.333
b
6.9 a
1.7 b
5.8 a
5.8 a
1a
1b
1.6 a
0.0
0.01
0.03
2.04 a 6.8 a
6.8 a
1.2 a
2.5 ab 1.058 a
0.1
2.4 a
6a
1.2 a
3.4 a
0.3
2.18 a 6.6 a
6.6 a
1.5 a
3.67 a 0.613 a
2.2bc
6.4 a
6.4 a
1.2 b
1.333
b
2.1 a
1.86 c
6.8 a
6.8 a
1b
1.67 b
1.333ab
2.18 bc
8.6 a
8.6 a
2a
2.6 b
0.676 b
2.72 a
8.4 a
8.4 a
1.2 b
4.6 a
1.626 ab
2.34 b
8.8 a
8.8 a
1.2 b
4.75 a
1.005 b
0.0
0.01
0.03
0.1
6a
0.3
Means with the same letter(s) are not significantly different using
Duncan Multiple Range Test.
1.556 a
Fig. 14: Effect of different cocentrations of NAA on plant height (cm) of in vitro
cultured plantlets of "Sarra"rose cultivar
A = 0.00mg/l
B = 0.01mg/l
C = 0.03mg/l
D = 0.10mg/l
E = 0.30mg/l
Fig. 15: Effect of different concentrations of NAA on number of roots of in
vitro cultured plantlets of "Sarra" rose cultivar
A = 0.00mg/l
B = 0.01mg/l
C = 0.03mg/l
D = 0.10mg/l
E = 0.30mg/l
Plate(7)
Table9:Effect of different concentrations of BA on in vitro growth
and development of rose axillary buds
Weeks
After
culture
plant
trea t.
mg/l
0.0
hight
(cm)
2.02 a
No.of
leaves
5.4 bc
No.of
nods
No.of
shoots
No.of
roots
Root
length
(cm)
5.4 b
1b
0.0
0.0
0.0
0.0
1.4 c
4.4 c
4.4 b
1b
0.3
2a
7a
7a
2.2 a 0.0
0.0
1.0
1.72 b 7.8 a 7.8 a 2.2 a 0.0
0.0
1.7 b
0.0
2.1 b
6.08 6.8
1.8
0.0
b
ab
ab
5.8 d 5.8 d 1.2 c 0.0
1.7 c
6.4 d
0.1
3.0
0.0
0.1
0.3
1.0
3.0
0.0
0.1
0.3
1.0
3.0
2.78 a 23.6
a
2.06 b 18 b
0.0
6.4 d
1.4 c
0.0
0.0
23.6
a
18 b
3.2
ab
4a
0.0
0.0
0.0
0.0
1.88
bc
2.2 b
11.8 11.8 2.8 b 0.0
c
c
6.4 d 6.4 d 1.2 c 1.33
b
0.0
1.92 c
10 c
10 c
1.2 c
5a
1.84 a
3.3 a
30 a
30.6 a
4b
0.0
0.0
2.4 ab
27 ab
27 ab
5.2 a
0.0
0.0
2.02 b
18.2 b
18.2 b
3.6 b
0.0
0.0
Means with the same letter(s) are not significantly different using
Duncan Multiple Range Test .
2.1 a
Fig. 16: Effect of different concentrations of BA on plant height (cm) of in
vitro cultured plantlets of "Sarra" rose cultivar
A = 0.00mg/l
B = 0.10mg/l
C = 0.30mg/l
D = 1.00mg/l
E = 3.00mg/l
Fig. 17: Effect of differrent concentrations of BA on number of shoots of in
vitro cultured plantlets of "Sarra" rose cultivar
A = 0.00mg/l
B = 0.10mg/l
C = 0.30mg/l
D = 1.00mg/l
E = 3.00mg/l
Plate (8)
4.7.3 BA + NAA:
Table (10) shows that there were significant differences between the different
concentrations of the two growth regulators on their effects on growth rates. The
lowest concentrations of both growth regulators (0.1 BA + 0.01 NAAmg/l) was the
best concentration for plant height (Fig.18) but when concentrations of both growth
regulators was increased plant height decreased. On the other hand number of leaves
(Fig. 19), number of nodes and number of shoots increased in highest concentration
(BA 3.0 + NAA 0.3 mg/l) of both growth regulators, while low concentrations and
medium free of growth regulators induced rooting. Plate (9) shows rose plantlet
growing on different combinations of BA + NAA concentrations.
4.8 The physical state of the medium:
As shown in table (11) there were no significant differences between all
parameters measured. (Fig. 20) shows the effect of physical state of the medium on
plant height, while (Fig. 21) shows the effect of the physical state of the medium on
number of leaves using agar or cotton. Plate (10) shows rose plantlets growing on the
two different physical states, cotton or agar.
4.9 The effect of darkness:
Four weeks of treatment with darkness resulted in a more significant increase
of plant height (Fig. 22) and significant decreases on number of leaves, number of
nodes (Fig.23), and number of shoots (Table 12).
Table 10:Effect of different combinations of BA+NAA
concentrations on
in vitro growth and development Of rose axillary buds.
plant
Weeks
After
culture
BA+NAA
mg/l
Hight
(cm)
No.of
nodes
No.of
shoots
No.of
roots
Root
length
(cm)
5.4 c
1b
0.0
0.0
10.6 a
1.8 ab
0.0
0.0
1.58 b 7 bc
7 bc
1.6 ab
0.0
0.0
1.38 b 7.2 abc
0.0
0.0
1.38 b 9.2 ab
7.2
1.2 b
abc
9.2 ab 2.2 a
0.0
0.0
2.1a
5.8 b
5.8 b
1.2 b
2a
1.17 a
2.02 ab
11.8 a
11.8 a
1.8 ab
1a
0.5 a
9 ab
1.8 ab
0.0
0.0
1.0+0.1
1.74
9 ab
bc
1.54 c 10 a
10 a
1.8 ab
0.0
0.0
3.0+0.3
1.42 c 12 a
12 a
2.4 a
0.0
0.0
0.0
0.1+0.01
0.3+0.03
1.0+0.1
3.0+0.3
0.0
0.1+0.01
0.3+0.03
2.02 a
No.of
leaves
1.92 a
5.4 c
10.6 a
0.0
0.1+0.01
2.2 a
6.4 b
6.4 b
1.2 b
1.33 a
2.1 a
2.16 a
12.6 a
12.6 a
1.6 ab
1a
0.8 a
10.6 ab
10.6 ab
1.8ab
0.0
0.0
0.3+0.03 1.9 ab
1.0+0.1
1.68 bc
12.6 a
12.6 a
2.2 a
0.0
0.0
3.0+0.3
1.5 c
15 a
15a
2.4 a
0.0
0.0
Means with the same letter(s) are not significantly different using
Duncan Multiple Range Test .
Fig. 18: Effect of different concentrations of BA+NAA on plant height (cm) of in vitro
cultured plantlets of "Sarra" rose cultivar
A = 0.00+0.00mg/l
B = 0.10+0.01mg/l
C = 0.30+0.03mg/l
D = 1.00+0.10mg/l
E = 3.00+0.30mg/l
Fig. 19: Effect of different concentrations of BA+NAA on number of leaves of
in vitro cultured plantlets of "Sarra" rose cultivar
A = 0.00+0.00mg/l
B = 0.10+0.01mg/l
C = 0.30+0.03mg/l
D = 1.00+0.10mg/l
E = 3.00+0.30mg/l
Plate(9)
Table 11:Effects of the physical stat e of the medium on in vitro
growth and development of rose axillary buds
Weeks
After
cultur
e
trea
t.
plant
hight
(cm)
1.24 a
A
No.of
leaves
4.6 a
No.of
nodes
No.of
shoots
No.of
roots
Root length
(cm)
4.6 a
1a
0.0
0.0
1.38 a
4.8 a
4.8 a
1a
0.0
0.0
1.46 a
10 a
10 a
2.2 a
0.0
0.0
1.54 a
10.6 a
10.6 a
2.4 a
0.0
0.0
2.3 a
15.8 a
15.8 a
2.8 a
0.0
0.0
2.45 a
16 a
16 a
2.75 a
0.0
0.0
B
A
B
A
B
A= control (agar)
B = cotton
Means with the same letter(s) are not significantly different using
Duncan Multiple Range Test
Fig. 20: Effects of the physical state of the medium on plant height (cm) of
in vitro cultured plantlets of "Sarra" rose cultivar
A = Agar
B = Cotton
Fig. 21: Effects of the physical stateof the medium on number of leaves
of in vitro cultured plantlets of "Sarra" rose cultivar
A = Agar
B = Cotton
Plate(10)
Weeks
After
culture
plant
trea t.
A
B
C
hight
(cm)
1.32 c
1.54
bc
1.68
abc
No.of
leaves
No.of
nodes
No.of
shoots
No.of
roots
Root
length
(cm)
12.4 a
2.4 a
0.0
0.0
13 a
13 a
2.2 ab
0.0
0.0
9.8 ab
9.8 ab 1.67 ab 0.0
0.0
12.4 a
1.82
7.2 ab
ab
2.04 a 6 b
D
E
7.2 ab 1.4 ab
0.0
0.0
6b
0.0
0.0
1b
Table 12:Effect of darkness on in vitro growth and development
of
rose axillary buds
A= control
B = 1 week after culture C= 2weeks after culture
D= 3 weeks after culture E =4 weeks after culture
Means with the same letter(s) are not significantly different using
Duncan Multiple Range Test
Fig. 22: Effect of darkness on plant height (cm) of in vitro
cultured plantlets of "Sarra"rose cultivar
Fig. 23: Effect of darkness on number of nodes of
in vitro cultured plantlets of "Sarra" rose cultivar
5- Discussion
5.1 Effect of different concentrations of MS salt mix on growth and
development of rose axillary buds:
there are various salt formulations in plant tissue culture developed for
various purposes, but the most popular in most tissue culture laboratories is Murshige
and Skoog salt, (1962). It is balanced ionically and contains high level of mineral
elements especially nitrogen (Masaad 1999). MS salt concentrations have a high
effect on plant growth. 1.0x MS concentration generally used gave the best results in
all parameters measured. The plantlets had the best appearance with the biggest and
greenest leaves, strong and longest shoots with out any deformity in all organs. The
highest concentration 4.0x had the poorest growth. Leaves and shoots were dwarf and
pale with a lot of burnt plant parts and callus formation. This poor growth may be
attributed to the toxic effect of salts at this concentration where the plants were
stressed. The lowest concentration 0.25x also resulted in weak growth and this may be
due to the lack of some essential mineral elements important for plant growth. This
result is similar to the findings of (Abd El Hameed 1999) and (Fernandez and Clark
1991) but does not agree with the results reported by (Masaad 1999). The differences
between the two species of plants may be the cause.
5.2 Effect of different concentration of NaH2PO4 salt on growth and development of rose axillary
buds:
Best growth and development of the plants was observed in the medium
containing the concentration 1.0x. leaves were big and dark green, shoots were strong
and healthy, and all other growth parameters were excellent, but the growth and
development decreased when very low or high concentrations (0.25x or 4.0x) were
used. This may be attributed to the lack of this salt at the low concentration and its
toxic effect at the high concentration. The plants were weak and pale with small and
burnt leaves and dwarf shoots. This result shows the importance of the addition and
direct effect of this salt on “Sarra” rose in vitro culture conferming previous reports
(Murashige, 1974).
5.3 Effect of energy sources (carbohydrates):
Plant tissues, organs, or cells cultured in vitro can not synthesize all essential
growth factors, and thus depend totally on what is supplied in the nutrient medium. A
source of energy and carbon in the form of sugars is usually added to the nutrient
media. The classical MS medium contained 3% sucrose as a source of energy and
carbon. However the optimum concentration and type of sugar varies with plant
species (Masaad 1999).
5.3.1 Effect of different concentrations of sucrose on growth and development
of rose axillary buds:
The results obtained showed that when increasing the concentration from
(0.75% to 3.0%) all growth rates increased, but they then started to decrease at high
concentrations. At 3.0% concentration plants were vigorously growing with dark
green leaves and strong shoots while at high concentrations growth was poor and
weak specially at (12.0%) concentration where callus formation was induced. These
results showed that sugar is very essential for plant growth but may be toxic at high
concentrations and adversely affect plants growth. These results agree with (Abd El
Hameed 1999) working with banana and (Hussein 2002) working with cidir who
found that 3.0% gave best shoot length and number of nodes but disagree with
(Masaad 1999) working with grapes. The differences in results may be attributed to
the variation in plant species.
5.3.2 Effect of different concentrations of glucose on growth and development
of rose axillary buds:
Sucrose can be substituted by glucose specially in monocots tissue culture.
Excellent results were obtained at 3.0% concentration where growth rates increased
from the lowest concentration used (0.75%) until the 3.0% concentration then
decreased to its lowest rate at the concentration (12.0%). These results are similar to
that of (Abd El Hameed 1999) but contradict the findings of (Ill – Whansul and
Korban 1998) who found that sucrose was better. This may be the result of different
media used in addition to differences in plants species. Masaad (1999) on the other
hand found the best results with glucose at low concentrations. This is referred to the
differences between plant species. Comparing all concentrations of glucose with there
similar sucrose ones we find that glucose concentrations were the best even at the
highest most toxic concentration (12.0%). This may be because glucose is a simple
sugar produced naturally by plants so it is easily absorbed and quickly assimilated
while sucrose need some time to hydroly to simple sugar before being absorbed by
plants.That is why glucose gives better results than sucrose. But the problem is that
glucose is more expensive than sucrose and it is not readily available as is sucrose. It
can be used for research purposes and as needed.
5.4 Effect of different concentrations of thiamie-HCL (Vitamin B1)
on growth and
development of rose axillary buds:
The result of the experiment proved that thimaimine-HCL is an essential
element for plant growth and development at all concentrations tested. It gave good
results and increased plant growth up to the highest concentration (1.6 mg/l) where
plant growth decreased meaning that very high concentrations may be toxic.
5.5 Effect of different concentrations of adenine sulphate (A/S) on growth and
development of rose axillary buds:
Adenine is one of the organic bases in nucleic acid of plant cell. It has been
used for the first time in plant tissue culture by Skoog and Tsue (1948). It is usually
added in the form of adenine sulphate – 2H2O because it is more soluble in water in this form. It is
categorized as growth regulator because its effects are similar to those of cytokinins especially Kinetin (Masaad 1999). The
results of this study proved that A/S had no significant differences between the different concentrations tested. However the
lowest concentration tested (20mg/l) significantly increased the number of leaves, nodes, and shoots while the concentration
in vitro cultured need lower
concentrations of A/S for optimum growth. Poor results were recorded at the highest
concentration (320 mg/l) meaning that higher concentrations of A/S were inhibitory.
(40mg/l) gave the best plant height. These results indicate that plants
5.6 Effect of different concentrations of myo-inositol on growth and development
of rose axillary buds:
Myo-inositol a sugar alcohol, has been considered in plant tissue culture as a
vitamin and it was never used as a source of energy or carbon source like other sugars
(Masaad 1999). In this study there were no significant differences between all
concentrations tested, however lower concentrations (50 mg/l) gave best plant height,
number of leaves, and nodes, while number of shoots was the best at the concentration
(200mg/l). this last result was similar to (Masaad 1999) who assured that high
concentration improved growth and development. This means that the responses to
may-inositol inclusion in culture media varied greatly from essential to beneficial and
can be added in low concentrations for “Sarra” rose cultivar to improve growth and
development of cultured axillary buds.
5.7 Effect of different concentrations of growth regulators on growth and
development of axillary buds:
Exogenously added auxin, and or cytokinins to in vitro cultures explants
result in a variety of responses which depend on type and concentration of hormone
added, plant species, type of explants and the purpose of culture initiation. Auxin
stimulate cell expansion while cytokinins promote cell division (Masaad 1999).
5.7.1 Naphthaline acetic acid (NAA):
Results showed that highest concentration (0.3 mg/l) was the optimum
concentration for number of leaves, nodes, and roots, but for plant height 0.1 mg/l was
better though differences were not significant compared to 0.3 mg/l.Best number of
shoots was obtained at (0.03 mg/l). Lower concentrations of NAA increased root
length while higher concentrations induce shorter roots, being shortest at (0.3 mg/l).
Best results for root length was recorded with media without growth regulators. These
results are similar to the reports of Arnold et al., 1995 who found that adding auxin to
the medium reduced roots length for all cultivars tested in his own experiment. All
culture with NAA rooted even at low concentrations proving that this growth
regulator is important for rooting of roses.
5.7.2 Benzyl adenine (BA):
Best results for plant height, number of leaves and nodes were obtained at
the concentration 0.3 mg/l but the highest number of shoots was obtained at 1.0 mg/l
with no significant differences when compared with 0.3 mg/l which follows it in its
positive effects. These results mean that cytokinins with moderate concentration give
best shoot growth because of its suppressive action on apical dominance of main
shoot meristem (Masaad 1999). BA is thus very important for rose shoot growth and
development.
5.7.3 Benzyl adenine + naphthalene acetic acid (BA + NAA):
Growth and development in plants is controlled by the internal interaction of
growth regulators. Auxins and cytokinin are the most important of these substances.
The interaction of both with the environment and the genetic mape up of the plant
control growth and developments in plants (Masaad 1999). Best results for number of
leaves, nodes and shoots were obtained at the highest concentration (3.0 BA + 0.3
NAA) of both growth regulators. Plant height was however was best on medium free
of both regulators. The experiment showed that the addition of growth regulators
inhibits plant height specially at high concentrations. In this experiment number of
leaves and plant height results are similar to the results of Masaad 1999 who found
that increasing BA + NAA concentrations increases number of leaves but decreases
plant height. The addition of BA +NAA inhibits rooting which were induced on
media free of or contains very low concentrations of these growth regulators. When
comparing BA + NAA results with BA or NAA added alone to the media we found
the following differences:
BA + NAA compared with BA added alone we found the best concentration
of BA which scored best results had better shoot growth and development than BA +
NAA combination best concentration .
When comparing NAA adding alone with BA + NAA combination in there
best concentration that scored best results we found that BA + NAA combination
failed to produce roots except at their lowest concentrations (BA 0.1 + NAA 0.01).
These results showed that adding growth regulators singly gave better results
than adding them in combination for “Sarra” rose cultivar taking the purpose of
culture (rooting or shoot growth in consideration).
5.8 Effect of the physical state of the medium on growth and development of rose
axillary buds:
Cotton fibers tested as an economical substitute for agar in tissue culture
because the cost of agar. Results showed that there were no significant differences in
all growth rates when using cotton compared with plants grown on agar solidified
medium. This means that agar has no direct effect on plant growth so it can be
substituted for by cotton or other substitutes. This result is similar to the previous
reports (Moreas – Cerdira 1995); (Rodriguez and Diaz – Sala 1991).
5.9 Effect of darkness:
Incubating culture plants in darkness did not improve plant growth except in
plant height which was affected by etiolation , but all other growth parameters
measured were reduced which means that there is no need for this treatment except
when there is an urgent need for a quick increase in plant height for other tissue
purposes.
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Appendix (1)
Appendix (1):Inorganic salt stocks of Murashige and Skoog(1962)
medium.
Chemical
Concentration(g/l)
Nitrate stock:
Ammonium nitrate,NH4NO3
Potassium nitrate,KNO3
165.0
190.0
Sulphate stock:
Magnesium sulfate,MgSO4.7H2O
Manganous sulfate,MnSO4.H2O
Zinc sulfate,ZnSO4.7H2O
Cupric sulfate,CuSO4.5H2O
37.0
1.69
0.86
0.0025
Halide stock:
Calcium chloride,CaCl2.2H2O
Potasium iodide,KI
Cobalt chloride,CoCl2.6H2O
PBMo stock:
Potasium phosphate,KH2PO4
Boric acid,H3BO3
Sodium
molybdate,Na2MoO4.2H2O
44.0
0.083
0.0025
17.0
0.620
0.025
NaFe EDTA stock:
Ferrous sulfate,FeSO2.7H2O
Ethylenediamineteraacetic
acid,disodium salt,
Na2EDTA
(Smith ,R. 1992).
(Smith ,R. 1992).
2.784
3.724
Appendix (2)
Medium
A
1.Sucrose
2.Thiamine- HCl
3.Myo-inositol
4.NAA
5.Cytokinins
6.Adenin sulphate
7.Agar
30.0
0.4
100
0.3
(BA)3.0
80
7000
B
30.0
0.4
100
0.2
Kinetin 2.0
80
7000
C
30.0
0.4
100
0.1
Kinetin 1.0
80
7000
Appendix (2)
Cemical composition of Murashige and Skoog Multiplication media
A, B, and C. All three media contain MS salt mix +NaH2PO4.2H2O